Construction of recombinant lentiviral vector containing human stem cell leukemia gene and its expression in interstitial cells of cajal

Chen Li; Biao Qian; Zhao Ni; Qinzhang Wang; Zixiong Wang; Luping Ma; Zhili Liu; Qiang Li; Xinmin Wang

doi:10.1515/biol-2020-0010

Article Open Access

Construction of recombinant lentiviral vector containing human stem cell leukemia gene and its expression in interstitial cells of cajal

Chen Li , Biao Qian , Zhao Ni , Qinzhang Wang , Zixiong Wang , Luping Ma , Zhili Liu , Qiang Li and Xinmin Wang

Published/Copyright: March 25, 2020

Published by

Become an author with De Gruyter Brill

Submit Manuscript Author Information Explore this Subject

From the journal Open Life Sciences Volume 15 Issue 1

Abstract

This study aims to construct recombinant lentiviral vectors containing the human stem cell leukemia (SCL) gene and investigate their in vitro transfection efficiency in Interstitial Cells of Cajal (ICC) of guinea pig bladders. In this study, the human SCL gene was successfully cloned, and the recombinant lentivirus GV287-SCL was successfully constructed. The titer of the recombinant lentivirus was 5 × 10⁸ TU /mL. After transfecting the ICCs with the lentiviral vector at different MOIs, the optimal MOI was determined to be 10.0, and the optimal transfection time was determined to be 3 days. The amplification product of the lentivirus transfection group was consistent with the target fragment, indicating that the SCL gene had been successfully introduced into ICCs. In conclusion, the recombinant lentiviral vector GV287-SCL was successfully constructed and transfected into the in vitro cultured ICCs. The successful expression of SCL in ICCs may provide an experimental basis for the in vivo transfection of the SCL gene.

Keywords: diabetic cystopathy; stem cell leukemia gene; lentiviral vectors; transfection; interstitial cells of Cajal

1 Introduction

Diabetic cystopathy (DCP) is one of the most common complications of late diabetes, and the incidence in diabetes patients is 19% ~ 84% [1, 2]. In late DCP, urinary tract infection and nephritis may occur and may develop into uremia [3, 4]. Many factors including decrease of nerve growth factor (NGF) levels, formation of oxidative free radicals, abnormal glucose metabolism, decrease of prostaglandin E2 (PGE2) levels, increase of mitochondrial reactive oxygen species (ROS), and reduction of the supply of blood vessels can lead to the occurrence of DCP [5, 6, 7]. However, the exact pathogenesis is still unclear. Recent studies have discovered a kind of specific cell type similar to the morphology of interstitial cells of Cajal (ICC) in the detrusor of humans, guinea pigs, and many other animals which is called bladder ICC [8, 9]. Studies confirm that spontaneous detrusor contractions completely regulated by ICCs occur, and thus ICCs may be possible bladder pacemaker cells [10, 11]. A tyrosine kinase receptor c-kit is present on the surface and cytoplasm of ICC cells and is considered to be a specific marker of ICC [12]. The SCL gene is an important regulatory gene upstream of c-kit, and it mainly acts on the gene promoter of c-kit, thereby regulating c-kit expression [13]. For DCP patients, ICCs continue to shrink or disappear due to the induction of hyperglycemia, which can damage the transmission functions of the stem cell factor (SCF)/growth factor receptor tyrosine kinase (c-kit) signaling pathway [14], lead to the reduction of bladder sensation, increase urine storage capacity, decrease detrusor contractility during urination, increase bladder residual urine and other discomforts, and result in bladder function becoming completely paralyzed or even failing [15].

There are two key receptors on ICCs, the specific protein receptor c-kit and its ligand stem cell factor (SCF). The specific binding of c-kit and SCF can start the SCF / c-kit signaling pathway [16]. Research has shown that the stem cell leukemia (SCL) gene is an upstream necessary regulator of c-kit which can increase the expression of c-kit and then promote the recovery of the ICC phenotype and function in DCP [17]. Therefore, the SCF/c-kit signaling pathway plays a key role in promoting ICC function reversal, which can guide the clinical treatment of DCP.

Recombinant lentiviral vectors containing human SCL gene can be used to infect ICC cells with morphological and functional damage to increase the number and promote the functional recovery of ICCs. In this study, recombinant lentiviral vectors containing the SCL gene were constructed using genetic engineering techniques and used to transfect in vitro cultured ICCs. The transfection efficiency and expression of the SCL gene were investigated. Our findings may provide new ideas for the further use of lentiviral vectors containing the human SCL gene in transfection experiments in vivo and DCP treatment.

2 Materials and Methods

2.1 Experimental animals

A total of 40 guinea pigs of either gender was used for the study. Their ages were 6-12 months and weight was 250-400 g. All guinea pigs were purchased from the Animal Experimental Center of Xinjiang Medical University.

Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experiments were conducted according to the ethical guidelines of the First Affiliated Hospital of Shihezi University.

2.2 Reagents

Competent E. coli DH5α, 293T cells, and GV287-EGFP vector were purchased from Genechem (Shanghai) Co. (Shanghai, China). DMEM medium, fetal bovine serum (FBS), and Lipofectamine 2000 transfection kit were purchased from Invitrogen Co. (Carlsbad, CA, US). RT-PCR kit was purchased from Fermentas Co. (Waltham, MA, US).

2.3 Construction and identification of lentiviral vectors containing the human SCL gene

The recombinant lentiviral plasmid containing the human SCL gene was constructed by the assistance of Genechem (Shanghai) Co. (Shanghai, China). Briefly, the human SCL gene was amplified by PCR. The primers were as follows: upstream 5’-GAGGATCCCCGGGTACCGGTCGCCACCAT-GACCGAGCGGCCGCCGAG-3’ and downstream 5’-TCAC-CATGGTGGCGACCGGCCGAGGGCCGGCTCCATC-3’.

The primers were synthesized by Genechem (Shanghai) Co. The PCR procedure was as follows: denaturing at 94oC for 5 min, 30 cycles of denaturation at 94oC for 30 s, annealing at 55oC for 30 s, extension at 72oC for 2 min, and a final extension at 72oC for 10 min. The PCR product was verified by agarose gel electrophoresis. Then, the PCR product was purified and inserted into the GV287-EGFP vector by In-Fusion converting enzyme (QIAGEN Co., Valencia, CA, USA) to construct recombinant plasmid GV287-EGFP/SCL. The correctly constructed recombinant plasmid GV287-EGFP/SCL was identified by sequencing.

2.4 Packaging of lentiviral vector and detection of virus titer

In a 1.5 mL sterile EP tube, 1.5 μg of packaging helper plasmids (Helper 1.0 and Helper 2.0, Genechem Co. Shanghai, China), 0.5 μg of expression plasmid, and 250 μL serum-free medium were added, mixed, and incubated at room temperature for 5 min. At the same time, 9 μL of liposomes was mixed with 250 μL serum-free medium and incubated at room temperature for 5 min. Then, the above DNA solution and liposome suspension were mixed and incubated at room temperature for 20 min. The 293T cell line was used as the packaging cell. The mixture of DNA and liposome was put into a 6-well plate and supplied with 1 mL complete medium. 293T cell suspension (1 ml) was put into the 6-well plate and incubated at 37oC in an atmosphere with CO₂ for 48-72 h. After that, the cells were centrifuged at 3000 r/min for 20 minutes, and the precipitate was filtered. The lentivirus stock solution was filtered with PVDF membrane. Virus-containing medium were grouped by 1: 100 dilutions and divided into standard solution and test solution. Both solutions were used for transfecting 293T cells simultaneously, and the virus titer was calculated by a fluorescent labeling method [8].

2.5 In vitro culture of bladder ICCs of guinea pigs

Four healthy guinea pigs were randomly selected and sacrificed by cervical dislocation. Their bladder tissues were removed in a sterile environment and cut into 1 mm³ pieces. The tissue pieces were digested with 1.0 mg/mL trypsin for 15 min at 37oC. DMEM medium (containing 1.0 mg/mL collagenase V, 100 U/mL penicillin, and 100 g/mL streptomycin) was added, and the tissues were further digested for 90 min at 37oC. Then, the digested tissues were filtered and centrifuged at 350 xg for 5 min. The supernatant was discarded, and the pellet was washed with 1% phosphate buffer solution (PBS). It was then centrifuged at 350 xg three times for 5 min each. Single cells were suspended by pipetting and then seeded in a 6-well plate. Low glucose DMEM medium containing 10% FBS and 1% double antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) were added to each well, and the plate was then cultured at 37oC in an incubator. After 24 hours, the cells were observed under an inverted microscope, and the non-adhered smooth muscle cells were removed. The adhered ICCs were transferred to culture dishes and identified by immunofluorescence after 72 hours of incubation.

2.6 Immunofluorescence

In a 6-well plate, a sterile slide was put into each well, and the ICC suspension was dropped on the center of the slide. About 1 mL medium was added to each well, and another 1 mL medium was added after 8 hours of incubation. After 48 hours of incubation, the slides were taken out. The primary (rat anti-mouse c-kit monoclonal antibody) and secondary antibody (FITC-labeled rabbit anti-rat antibody) were diluted with antibody diluent to reach their working concentration 1: 100. The procedure of the immunohistochemical test was as follows: briefly, the slides were incubated in 100% acetone at room temperature for 10 minutes. After washing with 0.01 M PBS three times (5 minutes each time), the endogenous peroxidase was inactivated with 0.03% H₂O₂ for 20 minutes at room temperature. Then, the slides were blocked with 1% FBS albumin for 30 minutes at room temperature. After blocking, the slides were incubated with primary antibody of rat anti-mouse c-kit monoclonal antibody (1: 100, Merck Millipore Co., Germany) in a wet box for 2 hours at room temperature and then for 48 hours at 4oC. After washing, FITC-labeled secondary antibody (rabbit anti-rat antibody, 1: 100, Sigma Co., St. Louis, MO, USA) was added and incubated in the dark for 60 minutes. Finally, the slides were sealed with cover glass using 50% glycerol and observed under a fluorescence microscope and a laser scanning confocal microscope.

2.7 Determination of the optimal MOI of the lentiviral vector containing human SCL gene into ICCs

According to the manual for lentiviral transfection (provided by Genechem Co.), ICCs were added with 5×10⁸ TU/mL lentiviral vectors containing the SCL gene at different MOIs (0.5, 1.0, 5.0, 10.0, 50.0, and 100.0), and 10 μg/mL polybrene was simultaneously added. After incubation for 8-12 h, the cells were observed under a microscope. After another 48 h incubation, the growth of ICCs was observed under a laser scanning confocal microscope, and the transfection efficiency was calculated [Transfection efficiency = (Number of cells expressing GFP/Total cell number) × 100%] to determine the optimal MOI.

2.8 Determination of the optimal transfection time of the lentiviral vector containing the human SCL gene into ICCs

ICCs were seeded in a 96-well plate at a concentration of 3×104~5×104/mL. The ICCs were divided into a normal control group, blank plasmid control group, and lentivirus transfection group using a random number table method. In the normal control group, 1% PBS was added. In the blank plasmid control group, blank lentivirus was added. In the lentivirus transfection group, the lentiviral vector containing human SCL gene was added at the optimal MOI together with 10 μg/mL polybrene. After 24 hours of incubation, the medium was removed, and the cells were washed with 1% PBS twice, and then 1 mL fresh DMEM medium containing 10% FBS was added for further incubation. The cells were observed using the laser scanning microscope at the 2, 3, and 5 d after transfection. A total of 20 continuous perpendicular to the horizontal views (about 0.77 mm²/view, 200 ×) were observed. The optimal transfection time was determined by observing the expression intensity of GFP.

2.9 RT-PCR

The total RNA of ICCs was extracted at 2, 3, and 5 d after transfection, and the expression of SCL mRNA was analyzed using RT-PCR. The primers of SCL and GAPDH were as follows:

SCL: Upstream: 5’-GAGGATCCCCGGTACCGGTCGC-CACCATGCGAGCGGCCGCCGAG-3’; Downstream: 5’-TCAC-CATGGTGGCGACCGACCGGCCGAGGGCCGGCTCCATC-3’.

GAPDH: Upstream: 5’-ACCACAGTCCATGCCATCAC-3’; Downstream: 5’-TCCACCACCCTGTTGCTGTA-3’.

The size of SCL is 1036 bp, while that of GADPH is 456 bp. The PCR reaction procedure was as follows: denaturing at 94oC for 5 min, 30 cycles of denaturation at 94oC for 30 s, annealing at 53oC for 30 s, and extension at 72oC for 1 min and a final extension at 72oC for 10 min. The products were detected with agarose gel electrophoresis. Gel imaging and gray intensity analysis were performed using a gel analysis system, and the expression of SCL mRNA in each group of ICCs was detected.

2.10 Statistical analysis

Data analysis was performed with the statistical software SPSS 17.0. The data was expressed as mean ± standard deviation (SD). ANOVA was used for the comparison among groups, and SNK-q test was used for the comparison between two groups. A P< 0.05 was considered to be statistically significant.

3 Results

3.1 Separation, culture and identification of guinea pig ICCs

To confirm whether the cultured cells were ICCs, c-kit antibody was used to identify the ICCs. After 24 hours of seeding, some spindle cells were observed with both sides and had obvious convex. Their nuclei were large, and there were some black glycogen particles in the cytoplasm. They had regular shapes, and most of them grew adherently (Figure 1A). After incubation for another 72 hours, the above cells were identified by immunofluorescence using the antibody of c-kit, which is an ICC-specific marker. The ICC-specific receptor tyrosine kinase (c-kit) was successfully expressed (Figure 1B). This result verified that the cultured cells were ICCs.

Figure 1

Identification of ICCs after GFP-SCL transfection. (A) After 2 days of incubation, adherent spindle cells with two projections on the two poles were observed under an inverted microscope (200×). (B) Tyrosine kinase (c-kit) receptor positive cells detected by immunofluorescence assays (200×).

3.2 The optimal MOI for the lentiviral transfection into ICCs

The GV287-SCL recombinant lentivirus vector was successfully constructed. The virus titer was 5 × 10⁸ TU/ mL. To determine the optimal MOI for the lentiviral vector containing the human SCL gene to transfect cells, transfection efficiency of different MOI was measured. After 48 h of transfection, green fluorescence was observed under the laser scanning confocal microscope. As shown in Figure 2, when MOI was 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0, the transfection efficiencies were 35.42% ± 0.12%, 58.04% ± 2.28%, 74.47% ± 3.22%, 85.62% ± 0.33%, 90.39% ± 0.67%, 90.77 ± 0.40%, respectively. When MOI ≥ 10.0, the transfection efficiencies all maintained above 85%, and there were statistically significant differences from those at MOI < 10.0 (P < 0.05, Table 1). When MOI ≤ 10.0, the cell growth was good and had no cell death. When MOI ≥ 50.0, although the transfection efficiencies were high, part of the cells were dead, and the cell activity decreased. Taking into consideration the transfection efficiency and cell activity at each MOI value, the optimal MOI for the lentiviral vector containing the human SCL gene was determined to be 10.0.

Figure 2

ICCs transfected with lentivirus at different MOIs for 48 h observed by an inverted phase contrast microscope (200×). The representative images at different MOI (MOI = 0.5; MOI = 1.0; MOI = 5.0; MOI = 10.0; MOI = 50.0; MOI = 100.0) were shown.

Table 1

Transfection efficiencies of the lentivirus into ICCs at different MOI values (mean ± SD, n=4).

MOI	Transfection efficiency (%)
0.5	35.42 ± 0.12^[a]
1.0	58.04 ± 2.28^[b]
5.0	74.47± 3.22^[c]
10.0	85.62 ± 0.33
50.0	90.39 ± 0.67^[d]
100.0	90.77 ± 0.40^[e]
F value	326.30
P value	<0.001

Note: MOI, multiplicity of infection

3.3 The optimal transfection time for the lentiviral vector into ICCs

To determine the optimal transfection time for the lentiviral vector containing the human SCL gene to ICCs, comparison of the green fluorescence in ICCs at different days was performed. The bladder ICC-like cells were transfected at the optimal MOI, and the transfection efficiencies were observed at 2 d, 3 d, and 5 d after transfection (Figure 3). After 3 days of transfection, the green fluorescence was the most obvious with the highest intensity, and after that the expression of GFP was weakened. After 3 d of lentiviral transfection, GFP expression in the ICCs was higher than in ICCs with 2d and 5d of transfection. Hence, the optimal transfection time for the lentiviral vector containing the human SCL gene in ICCs was 3 d.

Figure 3

The expression intensity of GFP in the lentivirus transfection group. The representative images at day 2, day 3, and day 5 are shown (200×).

3.4 The expression of SCL mRNA

To determine whether the SCL gene was successfully introduced into ICCs, RT-PCR was performed to determine the expression of mRNA in each group. The amplification product of the lentiviral group was consistent with the target fragment in size, while there was no expression of the specific band in the normal control group and blank plasmid control group (Figure 4). This indicated that the SCL gene was successfully introduced into ICCs.

Figure 4

The expression of SCL mRNA analyzed by agarose gel electrophoresis. Lane 1: Normal control group; Lane 2: Blank plasmid control group; Lane M: Marker; Lane 4 and 5: Lentivirus transfection groups.

4 Discussion

Studies [18, 19, 20] have indicated that ICCs can control the spontaneous excitement and contraction of the detrusor. It has been shown that bladder ICCs can spontaneously generate action potentials using patch clamp [21]. Blocking ICC action potentials significantly inhibits the contraction of detrusor cells [22]. Some researchers believe that ICCs are capable of neural information conduction and may serve as the bladder pacemaker [22, 23, 24]. However, ICC levels in the bladder detrusor of diabetic patients are low and show abnormal distribution or weakened spontaneous excitement, thus promoting the formation of DCP [25]. Related studies [26, 27] have found that the growth of ICCs is closely related to their specific tyrosine kinase receptor, c-kit. When the SCF/c-kit signaling pathway is successfully activated, the appearance and physiological characteristics of ICCs can be maintained [28]. SCL is a transcription factor that can regulate the expression and function of c-kit, but it cannot bind to DNA directly [29]. In the present study, the lentiviral vector containing the human SCL gene was constructed and transfected into in vitro cultured ICCs. After transfection, the expression of SCL in ICCs was enhanced. These results may provide relevant information to further explore gene therapy for DCP.

In this study, target cells were collected according to the adhesion time of bladder smooth muscle cells to bladder. After 24 h of incubation, ICCs were found to adhere to the culture plates, while bladder smooth muscle cells did not adhere. The non-adhered bladder muscle cells were filtered, and relatively pure bladder ICCs were obtained. Some characteristic spindle cells were observed under an inverted microscope. These cells had obvious convex branches on their sides, large nuclei, regular shapes, and were neatly adhered, while most of the non-adhered cells were oval, without projections on both poles and had overall flat shapes. The expression of the c-kit receptor was successful. Therefore, the cultured cells were confirmed to be bladder ICCs. The bladder ICCs obtained using this differential adherence method were of a relatively higher number and purer quality, but smooth muscle cells were still observed. This method is simple and time-cost effective and can provide enough cells for subsequent experiments.

Lentivirus is a commonly used gene vector adapted from the human immunodeficiency virus -1. It retains biological activity but has inactivated toxicity, low immunogenicity, and good biosafety [30]. Viral vectors can efficiently integrate target genes into host cells by gene recombination, unlikely to cause insertional mutagenesis [31]. The target genes can express sustainably and stably [32, 33]. Viral vectors have high transfection efficiencies for both dividing and non-dividing cells [34]. Thus, they are an ideal tool for gene transfection both in vitro and in vivo. In this study, lentivirus was packaged by a three plasmid system which included one recombinant virus plasmid encoding lentivirus and its two secondary packaging element vector plasmids. The encoding plasmid and the helper plasmids have cis-trans complementary functions. In addition, the structures of the secondary packaging plasmids were constructed by pHelper1.0 and pHelper2.0 vectors. This method is relatively simple, the lentivirus recombination rate and yield are acceptable, and the expression of the target gene is high. During the experiment, the supernatant rich in lentiviral particles was collected and concentrated and purified repeatedly to obtain high-titer lentiviral stock solution. The expression of the target gene was determined after transfecting 293T cells, and the virus titer was determined by fluorescence. The results showed that the titer of the lentivirus was 5 × 10⁸ TU /mL, which could meet the needs of most transfection experiments.

Although the toxic genes of lentivirus have been excluded, its biological risk is not completely lost. Lentivirus is a “suicide” virus with certain cytotoxicity [35, 36]. Studies have found that the transfection efficiency of lentivirus is closely related to its MOI, and high MOI can lead to some cell death [37, 38, 39, 40]. Therefore, a proper MOI is important. In the current experiment, we found that the lentiviral transfection efficiency in ICCs was best at MOI = 10.0, and cell activity was good with no cell death. Considering the transfection efficiency and growth state of the cells, the optimal MOI value was determined to be 10.0. GFP is a bioluminescent protein that can steadily emit green fluorescence under the excitation of blue light and has no cytotoxicity. It is now widely used in cell biology and biopsy as a molecular marker or reporter gene. In this study, GFP was used as a marker gene of the lentivirus because of its stable and clear fluorescence. Under a fluorescence microscope, real-time and in situ observation of living cells can be realized. Therefore, the transfection of the three plasmids into 293T cells to generate virus could be observed in real time, and accurate and intuitive results could be obtained. The ICCs were transfected with the lentivirus at the optimal MOI value and the expression of GFP was observed under a confocal microscope. The results showed that there was no GFP expressions in the normal control group and blank plasmid control group after 2 days of transfection. The GFP expression intensity in ICCs after 3 days of lentivirus transfection was higher than that after 2 days and 5 days, so the optimal transfection time for the lentivirus containing the human SCL gene into ICCs was 3 days. Comprehensively, for the transfection of lentivirus containing the human SCL gene into ICCs, the optimal MOI was 10.0, and the transfection efficiency, biological activity, and safety were highest after 72 hours of transfection. The results of RT-PCR indicated that the amplification product in the lentivirus transfection group had the same size as that of the target fragment, which was 1036 bp, while there was no expression of the specific band in the normal control group and blank plasmid control group. This suggested that the SCL gene was successfully introduced into ICCs.

There are some limitations to the present study. In vitro culture of cells is different from the body’s internal environment and might lose cell maintenance or auxiliary signals as well as interactions with other cells and proteins. In vitro experiments cannot completely replicate the in vivo situation, thus whether the transfection of DCP guinea pig bladder using a lentiviral vector containing the human SCL gene or other in vivo experiments can obtain the same results needs further study.

In conclusion, this study successfully constructed a recombinant lentiviral vector containing the human SCL gene by gene technology and molecular biological techniques. The titer of the lentivirus was 5 × 10⁸ TU/ mL. In vitro cultured ICCs were transfected with the lentiviral vector containing the human SCL gene, and SCL was successfully expressed in the ICCs. Our findings may provide a foundation for further in vivo transfection studies using the SCL gene.

qb2003_2000@126.com

Acknowledgements

This work was supported by National Natural Science Foundation [grant number 81360120].

Conflict of interest: Authors state no conflict of interest.

References

[1] Liu G, Daneshgari F. Diabetic bladder dysfunction. Chin Med J (Engl) 2014; 127: 1357-1364Search in Google Scholar

[2] Xu Y, Wang L, He J, Bi Y, Li M, Wang T et al. Prevalence and control of diabetes in Chinese adults. JAMA 2013; 310: 948-95910.1001/jama.2013.168118Search in Google Scholar PubMed

[3] Gomez CS, Kanagarajah P, Gousse AE. Bladder dysfunction in patients with diabetes. Current urology reports 2011; 12: 419-42610.1007/s11934-011-0214-0Search in Google Scholar PubMed

[4] Lin TL, Chen GD, Chen YC, Huang CN, Ng SC. Aging andrecurrent urinary tract infections are associated with bladder dysfunction in type 2 diabetes. Taiwan J Obstet Gynecol 2012; 51: 381-38610.1016/j.tjog.2012.07.011Search in Google Scholar PubMed

[5] Leiria LO, Monica FZ, Carvalho FD, Claudino MA, Franco-Penteado CF, Schenka A et al. Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels. Br J Pharmacol 2011; 163: 1276-128810.1111/j.1476-5381.2011.01311.xSearch in Google Scholar PubMed PubMed Central

[6] Li Y, Sun Y, Zhang Z, Feng X, Meng H, Li S et al. Cannabinoid receptors 1 and 2 are associated with bladder dysfunction in an experimental diabetic rat model. BJU Int 2013; 112: E143-15010.1111/bju.12172Search in Google Scholar PubMed

[7] Oberbach A, Jehmlich N, Schlichting N, Heinrich M, Lehmann S, Wirth H et al. Molecular fingerprint of high fat diet induced urinary bladder metabolic dysfunction in a rat model. PLoS One 2013; 8: e6663610.1371/journal.pone.0066636Search in Google Scholar PubMed PubMed Central

[8] Briggs Boedtkjer D, Rumessen J, Baandrup U, Skov Mikkelsen M, Telinius N, Pilegaard H et al. Identification of interstitial Cajal-like cells in the human thoracic duct. Cells Tissues Organs 2013; 197: 145-15810.1159/000342437Search in Google Scholar PubMed

[9] Wang JP, Ding GF, Wang QZ. Interstitial cells of Cajal mediate excitatory sympathetic neurotransmission in guinea pig prostate. Cell Tissue Res 2013; 352: 479-48610.1007/s00441-013-1572-3Search in Google Scholar PubMed

[10] Yin Z, Yang J. Bladder interstitial cells and pathophysiology. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2014; 39: 644-648Search in Google Scholar

[11] Zhang YG, X.W. H, W. Z, Ma WQ, Wu YS, Wu Q et al. Excitability change and significance of interstitial cells of Cajal in rat bladder with detrusor hyperreflexia. Journal of Clinical Urology 2015: 168-171Search in Google Scholar

[12] Wang L, Liang Y, Chen Q, et al. Identification and distribution of the interstitial cells of cajal in the abomasum of goats[J]. Cell Transplant, 2018, 27(2): 335-344.10.1177/0963689717722561Search in Google Scholar PubMed PubMed Central

[13] Lang R J, Tonta M A, Zoltkowski B Z, et al. Pyeloureteric peristalsis: role of atypical smooth muscle cells and interstitial cells of Cajal-like cells as pacemakers.[J]. J Physiol, 2006, 576(3):695-705.10.1113/jphysiol.2006.116855Search in Google Scholar PubMed PubMed Central

[14] Wang QZ, Shi-Qi JI, Zhu GD, Yun-Fei LI, Cai ZQ, Ying-Long LI et al. The morphologic changes and significance of interstitial cells of Cajal-like cells in the bladder of diabetic guinea pig. Chinese Journal of Clinical & Experimental Pathology 2009;Search in Google Scholar

[15] Liu YG, Wang QZ, Ding GF, Liu XJ, Zhou JM, Ying-Long LI et al. Effects of SCF on the expressions of c-kit mRNA and protein of interstitial cells of Cajal-like cells in the bladder of guinea pig in the high glucose environment. Chinese Journal of Clinical & Experimental Pathology 2011; 27: 282-285Search in Google Scholar

[16] Tan YY, Ji ZL, Zhao G, Jiang JR, Wang D, Wang JM. Decreased SCF/c-kit signaling pathway contributes to loss of interstitial cells of Cajal in gallstone disease. Int J Clin Exp Med 2014; 7: 4099-4106Search in Google Scholar

[17] Kart Y, Karakus OZ, Ates O, Hakguder G, Olguner M, Akgur FM. Altered expression of interstitial cells of Cajal in primary obstructive megaureter. J Pediatr Urol 2013; 9: 1028-103110.1016/j.jpurol.2013.02.003Search in Google Scholar PubMed

[18] Feng J, Liu Y, Gao J, Zhong Y, Shu Y, Meng M et al. Effect of exogenous stem cell factor on numbers of interstitial cells of Cajal and detrusor contraction in rats with underactive bladder. Journal of Third Military Medical University 2016;Search in Google Scholar

[19] Juszczak K, Maciukiewicz P, Drewa T, Thor PJ. Cajal–like interstitial cells as a novel target in detrusor overactivity treatment: true or myth? Central European Journal of Urology 2014; 66: 41310.5173/ceju.2013.04.art5Search in Google Scholar PubMed PubMed Central

[20] Kim SO, Song SH, Ahn KY, Kwon DD. Distribution of interstitial cells of cajal in menopausal rat urinary bladder showing detrusor overactivity. Int Neurourol J 2010; 14: 48-5310.5213/inj.2010.14.1.48Search in Google Scholar PubMed PubMed Central

[21] Kim BJ, Kim HW, Lee GS, Choi S, Jun JY, So I et al. Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine. J Ethnopharmacol 2013; 149: 668-67510.1016/j.jep.2013.07.017Search in Google Scholar PubMed

[22] Zuo DC, Choi S, Shahi PK, Kim MY, Park CG, Kim YD et al. Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-kappaB and MAP kinase. World J Gastroenterol 2013; 19: 1210-121810.3748/wjg.v19.i8.1210Search in Google Scholar PubMed PubMed Central

[23] Han S, Kim JS, Jung BK, Han SE, Nam JH, Kwon YK et al. Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice. Mol Cells 2012; 33: 243-24910.1007/s10059-012-2204-6Search in Google Scholar PubMed PubMed Central

[24] Lee J, Kim YD, Park CG, Kim MY, Chang IY, Zuo DC et al. Neurotensin modulates pacemaker activity in interstitial cells of Cajal from the mouse small intestine. Mol Cells 2012; 33: 509-51610.1007/s10059-012-2290-5Search in Google Scholar PubMed PubMed Central

[25] Sanders KM, Koh SD, Ward SM. Interstitial cells of cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 2006; 68: 307-34310.1146/annurev.physiol.68.040504.094718Search in Google Scholar PubMed

[26] Sung R, Kim YC, Yun HY, Choi W, Kim HS, Kim H et al. Interstitial cells of Cajal (ICC)-like-c-Kit positive cells are involved in gastritis and carcinogenesis in human stomach. Oncol Rep 2011; 26: 33-42Search in Google Scholar

[27] Wang XY, Chen JH, Li K, Zhu YF, Wright GW, Huizinga JD. Discrepancies between c-Kit positive and Ano1 positive ICC-SMP in the W/Wv and wild-type mouse colon; relationships with motor patterns and calcium transients. Neurogastroenterol Motil 2015; 26: 1298-131010.1111/nmo.12395Search in Google Scholar PubMed

[28] Ward SM, Sanders KM. Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks. Am J Physiol Gastrointest Liver Physiol 2001; 281: G602-61110.1152/ajpgi.2001.281.3.G602Search in Google Scholar PubMed

[29] Li F, Zhang L, Li C, Ni B, Wu Y, Huang Y et al. Adenovirus-mediated stem cell leukemia gene transfer induces rescue of interstitial cells of Cajal in ICC-loss mice. Int J Colorectal Dis 2010; 25: 557-56610.1007/s00384-010-0883-zSearch in Google Scholar PubMed

[30] Rusu MC, Pop F, Hostiuc S, Curca GC, Streinu-Cercel A. Extrahepatic and intrahepatic human portal interstitial Cajal cells. Anat Rec (Hoboken) 2011; 294: 1382-139210.1002/ar.21441Search in Google Scholar PubMed

[31] Ranzani M, Annunziato S, Calabria A, Brasca S, Benedicenti F, Gallina P et al. Lentiviral vector-based insertional mutagenesis identifies genes involved in the resistance to targeted anticancer therapies. Mol Ther 2014; 22: 2056-206810.1038/mt.2014.174Search in Google Scholar PubMed PubMed Central

[32] Xie B, Yu G, Ling S, Ma Z, Bai J. Establishment of MDCK cell lines which stably express FRT-LacZ gene by lentiviral system. Biotechnology 2016;Search in Google Scholar

[33] Zhang J, Wang D, Hu N, Wang Q, Yu S, Wang J. The construction and proliferative effects of a lentiviral vector capable of stably overexpressing SPINK1 gene in human pancreatic cancer AsPC-1 cell line. Tumour Biol 2016; 37: 5847-585510.1007/s13277-015-4405-zSearch in Google Scholar PubMed

[34] Pelascini LP, Janssen JM, Goncalves MA. Histone deacetylase inhibition activates transgene expression from integration-defective lentiviral vectors in dividing and non-dividing cells. Hum Gene Ther 2013; 24: 78-9610.1089/hum.2012.069Search in Google Scholar PubMed PubMed Central

[35] Hossain JA, Ystaas LR, Mrdalj J, Välk K, Riecken K, Fehse B et al. Lentiviral HSV‐Tk.007‐mediated suicide gene therapy is not toxic for normal brain cells. J Gene Med 2016; 18: 234-24310.1002/jgm.2895Search in Google Scholar PubMed

[36] Mohammadi Z, Shariati L, Khanahmad H, Kolahdouz M, Kianpoor F, Ghanbari JA et al. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy. Mol Biotechnol 2015; 57: 793-80010.1007/s12033-015-9872-3Search in Google Scholar PubMed

[37] Hu Y, Zhang Q, Zhang L, Tang XX, He HY. Basic fibroblast growth factor lentiviral vector-transfected sheep bone marrow mesenchymal stem cells and non-specific osteogenic gene expression. Mol Med Rep 2015; 12: 267-27210.3892/mmr.2015.3399Search in Google Scholar PubMed

[38] Huang GH, Yang XT, Chen K, Xing J, Zhu L, Hong-Jiang LI et al. Construction of lentiviral vector with over-expression of Porf-2 gene and transfection of neural cells. Journal of Shanghai Jiaotong University 2015; 87: 480Search in Google Scholar

[39] Jiao ZZ, Xue WJ, Tian XH, Li Y, Zheng J. Construction of VEGF165 lentivirus vectors and expression in adipose tissue-derived stem cells after transfection. Chin Pharm J 2016;Search in Google Scholar

[40] Wang X, Mani P, Sarkar DP, Roychowdhury N, Roychowdhury J. Ex vivo gene transfer into hepatocytes. Methods Mol Biol 2009; 481: 11710.1007/978-1-59745-201-4_11Search in Google Scholar PubMed

Received: 2018-08-29

Accepted: 2019-10-21

Published Online: 2020-03-25

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/biol-2020-0010

Keywords for this article

diabetic cystopathy; stem cell leukemia gene; lentiviral vectors; transfection; interstitial cells of Cajal

Creative Commons

BY 4.0