RNAi调控雄性小鼠睾丸uPA表达的研究

发布时间：2018-01-01 18:20

本文关键词：RNAi调控雄性小鼠睾丸uPA表达的研究　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：第一部分siRNA干扰TM4细胞uPA表达的研究目的：采用化学合成的小干扰RNA (siRNA)转染小鼠睾丸支持细胞株TM4细胞,观察uPA表达的变化,筛选uPA-siRNA的有效干扰序列,为后续构建慢病毒介导的受Tet调控的uPA干扰载体提供实验依据。方法：在验证TM4细胞有内源性uPA表达的基础上,针对小鼠uPA mRNA靶序列设计三段不同的siRNA序列(si-uPA1,si-uPA2, si-uPA3),通过荧光标记的siRNA (Cy3-siRNA)确定有效转染浓度后,在阳离子脂质体Lipofectamine2000介导下分别将三段si-uPA序列瞬时转染TM4细胞,设立空白对照组(未转染)、试剂对照组(加入Lipofectamine2000)和阴性对照组(转染阴性siRNA, siRNA-scramble)。采用实时荧光定量PCR技术(qRT-PCR)和Western-Blot技术检测转染后各组细胞uPA mRNA和uPA蛋白的表达情况,分析比较uPA表达的变化,确定si-uPA的有效干扰序列。针对该序列,设定不同浓度(30 nM、50 nM和100 nM)的siRNA转染TM4细胞,分别作用24 h、48 h和72 h后,qRT-PCR测定uPA mRNA改变,观察siRNA对TM4细胞uPA表达的影响。结果：TM4细胞中有内源性uPA表达。20 nM的Cy3-siRNA转染TM4细胞后仅见微弱的Cy3红色荧光,随着转染浓度的增加,荧光浓度逐渐增强,表达红色荧光的TM4细胞增多,以50 nM和100 nM最为明显,选定50 nM作为siRNA的有效转染浓度。三种si-uPA转染TM4细胞后,uPA表达量均较空白对照组明显下降(P0.05),以si-uPA1作用最为明显,而试剂对照组uPA mRNA表达无明显改变。用si-uPAl转染TM4细胞进行时效量效实验,结果显示,转染24 h后,转染组细胞uPA mRNA的表达均较对照组显著降低,其中100 nM组抑制效果最为明显,抑制率达到70%,抑制效果强于30 nM组和50 nM组(P0.05)；随转染时间的延长,uPA mRNA表达持续降低,转染72 h后,三组细胞uPA mRNA表达量分别为对照组的53.9%、35.3%和27.7%,差异具有统计学意义(P0.05)。结论：本部分成功筛选出针对uPA mRNA靶序列的有效干扰序列si-uPA1,转染TM4细胞后,在24 h即可抑制细胞uPA mRNA的表达,抑制效应持续至72 h；同一浓度的si-uPA1在不同时间点内的抑制效应无显著性差异；同一时间点内,抑制效应随转染浓度的增加而增强,表现出良好的量效关系。第二部分慢病毒介导的可诱导RNAi体系的建立目的：在第一部分实验基础上构建慢病毒介导的含有Tet调控元件的uPA干扰载体,进行病毒包装和滴度测定后,感染TM4细胞,利用抗性筛选获得TM4双稳转细胞株,为后续Dox调控提供前提条件。方法：根据第一部分实验获得的uPA-siRNA的有效干扰序列si-uPA1,设计合成uPA-shRNA oligo,退火成双链,插入入门载体pENTR/H1/TO中,转化感受态细胞,获取入门克隆。利用Gateway技术的LR重组反应,将入门克隆中的Hl/TO-RNA干扰序列转入慢病毒表达载体pLenti4-BLOCK-iT-DEST中,转化感受态细胞,提取质粒,测序验证,构建针对uPA的慢病毒干扰载体pLenti4-sh。将慢病毒表达载体pLenti4-sh和含有四环素抑制子(tetracycline repressor, TetR)的慢病毒载体pLenti6/TR分别转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,运用qRT-PCR技术测定病毒滴度。用不同浓度的抗生素以及带EGFP的慢病毒原液(Lentil-EGFP)感染TM4细胞,选择抗生素的最佳筛选浓度和病毒感染的感染复数(multiplicity of infection, MOI)值。在此基础上,将pLenti6/TR慢病毒液感染TM4细胞,用Blasticidin筛选阳性细胞,qRT-PCR检测TetR的表达,建立TetR稳转细胞株TM4-Lenti6/TR;将pLenti4-sh慢病毒液感染TM4-Lenti6/TR,用Blasticidin和Zeocin共同筛选阳性细胞,qRT-PCR检测TetR和抗性基因的表达,建立双稳转细胞株TM4-Lenti6/TR Lenti4-sh. 结果：针对uPA靶序列构建的慢病毒干扰载体为pLenti4-sh,经测序验证,序列比对完全正确。慢病毒包装后,qRT-PCR测定Lenti6/TR病毒的物理滴度为9.8×109vp/ ml, Lenti4-sh病毒的物理滴度为1.72×1010vp/ml。不同浓度抗生素作用TM4细胞后,根据细胞生长状况观察,选择Blasticidin最佳筛选浓度为1μg/ml, Zeocin的最佳筛选浓度为600μg/ml.随着感染TM4细胞的Lentil-EGFP MOI值的增大,表达EGFP的TM4细胞增多,荧光亮度增强。因而将慢病毒感染TM4细胞的MOI设立为100。根据2-ΔΔCt的方法相对定量,TM4-Lenti6/TR细胞中TetR基因的表达量为正常TM4细胞的27301.01倍,而TM4-Lenti6/TR Lenti4-sh细胞中TetR基因和抗性基因表达量分别正常TM4细胞的406623.29倍和51212760.84倍。结论：成功构建了慢病毒介导的含有Tet调控元件的uPA干扰载体。经TM4细胞感染和抗性筛选后,建立了TetR稳转细胞株TM4-Lenti6/TR和双稳转细胞株TM4-Lenti6/TR Lenti4-sh. 第三部分可诱导的RNAi调控uPA表达的实验研究目的：利用第二部分实验中获得的稳转细胞株和携带uPA干扰载体的慢病毒液,构建体外细胞培养体系和小鼠在体动物模型,通过Dox给药,观察双稳转细胞株和小鼠睾丸组织uPA mRNA和蛋白表达情况以及uPA酶活性的改变,评估Dox对uPA的诱导效应。方法：通过MTT实验检测不同浓度Dox对TM4细胞的毒性,筛选Dox作用安全剂量。在此基础上,将第二部分获得的双稳转细胞株TM4-Lenti6/TR Lenti4-sh进行体外培养,以TetR稳转细胞株TM4-Lenti6/TR和TM4细胞作为对照,通过Dox诱导,在24 h、48 h、72 h观察各组细胞uPA mRNA和蛋白表达的抑制情况以及细胞培养液中uPA酶活性的改变,评估Dox的体外诱导效应。在Dox作用72 h后,移除Dox,观察移除24 h、48 h、72 h后uPA mRNA和蛋白表达的恢复情况。将慢病毒液Lenti4-sh和Lenti6/TR经皮睾丸注射,构建小鼠实验模型,以注射Lenti6/TR慢病毒液和生理盐水的小鼠作为对照,通过Dox灌胃进行体内诱导,在Dox作用24 h、48 h、72 h和1周后观察各组小鼠睾丸组织uPA mRNA和蛋白的表达情况以及睾丸组织匀浆液uPA酶活性变化。结果：MTT实验筛选得出Dox作用于TM4细胞的安全浓度在5μg/ml以下。以1μg/ml的Dox对TM4-TetRshuPA、TM4-TetR和TM4细胞进行体外诱导,结果显示,后两组细胞uPA表达在诱导组和非诱导组间无明显差异,而TM4-TetRshuPA双稳转细胞组的uPA mRNA在Dox诱导24 h后即较非诱导组有明显下降,随诱导时间的延长,抑制作用更为明显,作用72 h时,诱导组表达量仅为非诱导组的35%。uPA蛋白表达变化较mRNA出现略晚,在Dox诱导72 h后,蛋白表达较非诱导组显著降低。S-2251发色底物法检测结果示,双稳转细胞诱导组uPA酶活性在72 h时明显降低,为非诱导组的65.1%。随着Dox从细胞培养液的移除,uPA表达逐渐恢复,移除48 h后,uPAmRNA和蛋白表达水平与非诱导组无显著性差异。雄性小鼠经皮睾丸内注射慢病毒载体后,以0.75 mg/ml的Dox进行诱导,结果表明,TetRshuPA小鼠在Dox作用24 h后,uPA mRNA水平较非诱导组以及对照组明显降低,随Dox作用时间的延长,uPA mRNA的表达逐渐减少,Dox作用1周后,抑制作用最为明显,uPA mRNA表达水平与诱导24 h比较差异具有统计学意义。uPA蛋白与mRNA表达情况略有不同,在Dox作用的72 h内,TetRshuPA小鼠睾丸uPA蛋白与非诱导组表达水平相接近,而在Dox作用1周后,uPA蛋白表达水平在诱导组出现了明显下降,与非诱导组有显著差异。结论：Dox对细胞培养体系和小鼠睾丸组织内的uPA表达均有调控作用,其抑制uPA表达在体外体系可维持至72 h,移除Dox后,uPA表达水平在48 h内逐渐恢复。小鼠在体模型中,Dox诱导可以下调睾丸组织中uPA表达水平以及匀浆液uPA酶活性,抑制作用至诱导1周最为明显。第四部分调控uPA表达影响雄性小鼠生育力的实验研究目的：通过第三部分动物体内诱导实验,观察睾丸uPA表达受到可诱导的RNAi调控后,雄性小鼠生育力、精子功能的改变,分析uPA影响雄性小鼠生育力的可能机制。方法：慢病毒液经皮睾丸注射构建雄性小鼠动物模型,Dox灌胃1周后行交配实验,观察雄鼠交配率、交配雌鼠妊娠率、平均胚胎数和黄体数。解剖雄鼠,测定附睾精子活动百分率和存活率,低渗肿胀实验观察精子膜功能。小鼠睾丸附睾取材,光镜和透射电镜观察组织病理和超微结构改变,并观察动物生殖系统外其他组织器官病理变化。分别在Dox停药2周、4周和8周再次与雌鼠交配,观察雄鼠生育力和精子功能的恢复情况。结果：Dox给药1周后各组小鼠交配率无明显差异。TetRshuPA小鼠诱导组雌鼠妊娠率和平均胎数较非诱导组和空白对照组显著降低,差异具有统计学意义(P0.05),平均黄体数无显著性差异。停药2周后,TetRshuPA诱导组雌鼠妊娠率和平均胎数明显增加,至停药8周时,雌鼠妊娠率和平均胎数与非诱导组和空白对照组水平相当。 Dox诱导后,各组小鼠的精子存活率和精子低渗肿胀率均无明显差异。TetRshuPA小鼠活动精子百分率较非诱导组和空白对照组明显下降(P0.05)。随Dox停药时间的延长,TetRshuPA诱导组小鼠精子活力逐渐恢复,至停药8周时,诱导组小鼠精子活力恢复至空白对照组水平。 Dox用药后,各组小鼠睾丸附睾组织结构和超微结构未见明显病理改变。实验组小鼠其他组织器官未见病理性结构损伤。结论：Dox体内诱导睾丸uPA表达下调后不影响雄鼠的交配率及雌鼠黄体数,交配雌鼠的妊娠率和平均胎仔数下降。uPA的下调不影响精子存活率和精子膜功能,但可导致雄鼠精子活力降低。雄鼠生育力和精子活力在Dox停药8周后可完全恢复。uPA下调对雄鼠睾丸、附睾病理结构和超微结构无明显病理影响,雄鼠的心、肝、脾、肺、肾等主要脏器形态未见明显损伤性改变。
[Abstract]:Study on the expression of uPA in TM4 cells by siRNA interference in part one
Aim: to transfect TM4 cells from mouse testicular Sertoli cell line with chemically synthesized small interfering RNA (siRNA), observe the change of uPA expression, and screen the effective interference sequence of uPA-siRNA, so as to provide experimental evidence for subsequent construction of lentivirus mediated uPA interference vector regulated by Tet.
Methods: Based on the expression of endogenous uPA in the verification of TM4 cells, three different siRNA sequences were designed according to mouse uPA mRNA target sequence (si-uPA1, si-uPA2, si-uPA3, siRNA) by fluorescent labeling (Cy3-siRNA) to determine the effective concentration after transfection, the cationic liposome mediated by Lipofectamine2000 respectively under three si-uPA instantaneous sequence transfection of TM4 cells, a blank control group (without transfection reagent), control group (Lipofectamine2000) and negative control group (transfected with siRNA, siRNA-scramble). Using the real-time quantitative PCR (qRT-PCR) expression of mRNA cells uPA and uPA protein was detected and Western-Blot technology, analysis and comparison of the changes of the expression of uPA sure, effective si-uPA interference sequence. The sequence set different concentrations (30 nM, 50 nM and 100 nM) of siRNA were transfected into TM4 cells, respectively, 24 h, 48 h and 72 h, qRT-PCR The changes of uPA mRNA were measured and the effect of siRNA on the expression of uPA in TM4 cells was observed.
Results: the red fluorescence in TM4 cells the expression of endogenous uPA.20 nM Cy3-siRNA transfected TM4 cells were faint Cy3, with the increase of transfection concentration, the fluorescence concentration gradually increased, the expression of red fluorescence of TM4 cells increased to 50 nM and 100 nM was the most obvious, 50 selected nM as effective transfection concentration of siRNA. Three si-uPA after transfection into TM4 cells, the expression levels of uPA were significantly decreased compared with control group (P0.05), the effect of si-uPA1 was the most obvious, and the reagent control group uPA mRNA showed no significant change. TM4 cells were transfected with si-uPAl prescription dose effect experiment, results showed that 24 h after transfection, the expression of uPA in transfected cells mRNA was significantly lower than control group, of which 100 inhibitory effect of nM group was the most obvious, the inhibition rate reached 70%, the inhibitory effect is stronger than that of 30 nM group and 50 nM group (P0.05); with the extension of the time of transfection, the expression of mRNA uPA decreased 72 h after transfection, three group The expression of uPA mRNA in the cell was 53.9%, 35.3% and 27.7% in the control group, and the difference was statistically significant (P0.05).
Conclusion: we successfully screened effective si-uPA1 interference sequence for uPA mRNA target sequence, after transfection into TM4 cells, inhibiting the expression of uPA mRNA cells in 24 h and the inhibitory effect lasted for 72 h; the inhibitory effect of the same concentration of si-uPA1 at different time points in no significant difference; at the same time point in the inhibitory effect increased with the concentration of transfection showed that the dose-response relationship.
The second part of the inducible RNAi system mediated by lentivirus
Objective: Based on the first part of the experiment, we constructed lentivirus mediated uPA interference vector containing Tet regulatory elements. After virus packaging and titer determination, TM4 cells were infected, and TM4 bistable cell lines were obtained by resistance screening, providing a precondition for subsequent Dox regulation.
Methods: according to the effective interference sequence si-uPA1 to obtain the first part of the experiment of uPA-siRNA, the design and synthesis of uPA-shRNA oligo, the double strand annealing, inserted into the entry vector pENTR/H1/TO and transformed into competent cells, get started cloning. Recombinant LR reaction by using Gateway technology, the Hl/TO-RNA interference Sequence Cloning of the lentivirus into door expression vector pLenti4-BLOCK-iT-DEST. Transformation competent cells, Plasmid Extraction, sequencing, to construct uPA lentivirus vector pLenti4-sh. Lentivirus Expression Vector pLenti4-sh containing tetracycline repressor (tetracycline repressor, TetR) of the lentiviral vector pLenti6/TR were transfected into 293T cells, virus, virus stock solution was collected and concentrated by ultracentrifugation, the virus titer was determined by using qRT-PCR technology with different concentrations of antibiotics and EGFP lentiviral supernatant (Lentil-EGFP) infected TM4 cells, selection The best choice of antibiotic screening concentration and virus infection the multiplicity of infection (multiplicity of, infection, MOI). On this basis, the liquid pLenti6/TR lentiviral infection of TM4 cells, cells were screened by Blasticidin, to detect the expression of qRT-PCR TetR, the establishment of TetR stable cell strain TM4-Lenti6/TR; the pLenti4-sh lentivirus infection liquid TM4-Lenti6/TR, screening the positive cells with Blasticidin and Zeocin, to detect the expression of TetR and qRT-PCR genes, a double stable cell strain TM4-Lenti6/TR Lenti4-sh.
Results: the lentivirus vector uPA to construct the target sequence of pLenti4-sh, confirmed by sequencing, sequence alignment is correct. Lentivirus packaging, physical titers of qRT-PCR for detection of Lenti6/TR virus was 9.8 * 109vp/ ml, physical titers of Lenti4-sh virus was 1.72 * 1010vp/ml. with different concentrations of antibiotics in TM4 cells, according to the observation of cell growth select the best condition, Blasticidin screening concentration was 1 g/ml, the best Zeocin screening concentration was 600 g/ml. with the increase of TM4 cells infected with Lentil-EGFP MOI, the expression of EGFP TM4 cells increased, the fluorescence brightness is enhanced. The establishment will therefore slow virus infection of TM4 cell MOI 100. 2- Delta Delta Ct method according to the relative the quantitative expression of TetR gene in TM4-Lenti6/TR cells was 27301.01 times higher than that of normal TM4 cells, Lenti4-sh cells and TetR TM4-Lenti6/TR gene and resistance gene expression were normal TM The 4 cells were 406623.29 times and 51212760.84 times more than that of the cells.
Conclusion: lentivirus mediated uPA interference vector containing Tet regulatory elements has been successfully constructed. After TM4 cell infection and resistance screening, TetR stable transformation cell line TM4-Lenti6/TR and bistable cell line TM4-Lenti6/TR Lenti4-sh. were established.
Experimental study on the regulation of the expression of uPA by third inducible RNAi
Objective: the second part of the experiment of stabletransfection cell lines and uPA interference vector of lentivirus carrying liquid, in vitro cell culture system and animal model in mice, administered by Dox, observe the bistable cell lines and mouse testis tissue uPA mRNA and protein expression and uPA activity induced. Evaluation of the effects of Dox on uPA.
Methods: the MTT assay with different concentrations of Dox on TM4 cell toxicity screening Dox safe dose. On this basis, the second part of the double stable cell strain TM4-Lenti6/TR Lenti4-sh were cultured in vitro with TetR stably transfected cell lines TM4-Lenti6/TR and TM4 cells as control, induced by Dox, 24 h, 48 h uPA, the enzyme activity in the liquid to change the expression of H 72 cells were observed uPA mRNA and protein inhibition and cell culture, induced effect assessment of Dox in vitro. The effects of Dox on 72 h after removal of Dox, H 48 h 24 were removed, and the recovery of the expression of uPA protein and mRNA 72 h will slow. The virus was Lenti4-sh and Lenti6/TR percutaneous testicular injection, the murine experimental model, using liquid and saline injection Lenti6/TR lentivirus were used as controls, by gavage of Dox in vivo induced in Dox, 24 h, 48 h, 72 and h were observed after 1 weeks The expression of uPA mRNA and protein in mouse testis and the changes of uPA enzyme activity in the homogenate fluid of the testis.
Results: the safe concentration of MTT experimental screening that the effect of Dox on TM4 cells in 5 g/ml to 1 g/ml. The Dox of TM4-TetRshuPA, TM4-TetR and TM4 cells were induced in vitro. The results showed that the two groups after the expression of uPA in induced group and non induced no significant differences between groups, and TM4-TetRshuPA double stabletransfection cell uPA mRNA in the Dox group after 24 h induction than that of non induced group decreased significantly, with the induction time. The inhibitory effect is more obvious, the role of 72 h, the induction group expression is only non induction group 35%.uPA protein expression than mRNA slightly later, induced by Dox after 72 h protein the expression of a non induced group decreased significantly.S-2251 chromogenic substrate assay showed that the bistable cell induced group uPA activity decreased significantly at 72 h, for the non induced group 65.1%. with Dox from the cell culture medium was removed, the expression of uPA recovered gradually, removed after 48 h, and uPAmRNA There was no significant difference between the protein expression level and the non induced group.
Male mice testis by percutaneous injection of lentiviral vectors, with 0.75 mg/ml of Dox were induced by TetRshuPA in mice. The results showed that Dox 24 h, uPA and mRNA levels than non induced group and the control group decreased significantly, with the prolongation of Dox action time, the expression of uPA mRNA gradually decreased after 1 weeks Dox effect of inhibition is most obvious, with differences between the expression level of uPA mRNA and H.UPA expression induced by 24 significant protein and mRNA is slightly different, in the role of Dox 72 h, TetRshuPA mouse uPA protein and non induced group expression level is close to that in Dox after 1 weeks, the expression level of uPA protein in the induction group declined significantly, and the non induced group were significantly different.
Conclusion: the expression of Dox had regulation system and testicular tissue in mice uPA of cell culture, the expression of uPA in vitro system can be maintained to 72 h, removal of Dox, uPA expression was gradually recovered in 48 h mice. In vivo, Dox can induce uPA enzyme activity level and homogenate uPA expression downregulation of testis tissue, inhibition to 1 weeks of induction is the most obvious.
Experimental study on the effect of the fourth part of the regulation of uPA expression on the fertility of male mice
Objective: through third animal induction experiments, we observed the change of uPA expression in male testis and the changes of fertility and sperm function of male mice after induced RNAi regulation, and analyzed the possible mechanism of uPA affecting fertility in male mice.
Methods: lentiviral liquid percutaneous testicular injection of male mice to build animal model of Dox by gavage for 1 weeks after mating experiment, observation of male mating rate, mating female pregnancy rate, the average number of embryos and the number of corpus luteum. Anatomy of the male rats, determination of epididymal sperm activity rate and survival rate, to observe the function of sperm membrane swelling experiments of low infiltration. Mouse testis and epididymis were changed with light microscope and transmission electron microscope to observe the pathological and ultrastructural observation of animal reproductive system, and other organs. The pathological changes in the Dox withdrawal for 2 weeks, 4 weeks and 8 weeks again mating with female mice, to observe the recovery of fertility and sperm function in male rats.
Results: Dox administration 1 weeks after the mice mating was no significant difference between the number of.TetRshuPA mice than non induced group and blank control group group significantly decreased pregnancy rate and average embryo, the difference was statistically significant (P0.05), no significant difference in the average number of corpus luteum. After 2 weeks of discontinuation, TetRshuPA induced group the pregnancy rate and the average number of fetal rats increased significantly, to stop drug 8 weeks, pregnancy rate and the average number of births and non induced group and blank control group level.
After Dox induction, mice sperm survival rate and sperm hypotonic swelling rate were no significant difference between the percentage of motile sperm in.TetRshuPA mice than in the non induced group and blank control group decreased significantly (P0.05). With the extension of the Dox withdrawal time, TetRshuPA induced mice sperm motility recovered gradually to 8 weeks of discontinuation, induction mice sperm motility recovered to the control group.
After administration of Dox, there was no significant pathological change in the structure and ultrastructure of testis and epididymis in each group. There was no pathological structural damage in other tissues and organs of mice in the experimental group.
Conclusion: Dox in vivo induced testicular uPA expression does not affect the mating rate of male rats and luteal number of female rats, the pregnancy rate of mated female rats and decreased the average litter size does not affect the downregulation of.UPA function of sperm survival rate and sperm membrane, but can lead to reduced sperm activity of male mouse. Fertility and sperm motility in male rats 8 weeks after Dox withdrawal can be fully restored by testis of male rats.UPA, epididymis pathological structure and ultrastructure of male rats had no obvious pathological effect, heart, liver, spleen, lung, kidney and other organ damage there was no obvious morphological change.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346

【参考文献】