本文關(guān)鍵詞：RNAi調(diào)控雄性小鼠睪丸uPA表達的研究　出處：《華中科技大學》2011年博士論文　論文類型：學位論文

  更多相關(guān)文章： uPA RNA干擾 siRNA TM4細胞 基因表達 慢病毒載體 四環(huán)素抑制子 抗性篩選 穩(wěn)轉(zhuǎn)細胞株 TM4細胞 強力霉素 誘導(dǎo)表達 小鼠 睪丸 妊娠率 精子活力 精子存活率 低滲腫脹 超微結(jié)構(gòu)

【摘要】：第一部分siRNA干擾TM4細胞uPA表達的研究 目的：采用化學合成的小干擾RNA (siRNA)轉(zhuǎn)染小鼠睪丸支持細胞株TM4細胞,觀察uPA表達的變化,篩選uPA-siRNA的有效干擾序列,為后續(xù)構(gòu)建慢病毒介導(dǎo)的受Tet調(diào)控的uPA干擾載體提供實驗依據(jù)。 方法：在驗證TM4細胞有內(nèi)源性uPA表達的基礎(chǔ)上,針對小鼠uPA mRNA靶序列設(shè)計三段不同的siRNA序列(si-uPA1,si-uPA2, si-uPA3),通過熒光標記的siRNA (Cy3-siRNA)確定有效轉(zhuǎn)染濃度后,在陽離子脂質(zhì)體Lipofectamine2000介導(dǎo)下分別將三段si-uPA序列瞬時轉(zhuǎn)染TM4細胞,設(shè)立空白對照組(未轉(zhuǎn)染)、試劑對照組(加入Lipofectamine2000)和陰性對照組(轉(zhuǎn)染陰性siRNA, siRNA-scramble)。采用實時熒光定量PCR技術(shù)(qRT-PCR)和Western-Blot技術(shù)檢測轉(zhuǎn)染后各組細胞uPA mRNA和uPA蛋白的表達情況,分析比較uPA表達的變化,確定si-uPA的有效干擾序列。針對該序列,設(shè)定不同濃度(30 nM、50 nM和100 nM)的siRNA轉(zhuǎn)染TM4細胞,分別作用24 h、48 h和72 h后,qRT-PCR測定uPA mRNA改變,觀察siRNA對TM4細胞uPA表達的影響。 結(jié)果：TM4細胞中有內(nèi)源性uPA表達。20 nM的Cy3-siRNA轉(zhuǎn)染TM4細胞后僅見微弱的Cy3紅色熒光,隨著轉(zhuǎn)染濃度的增加,熒光濃度逐漸增強,表達紅色熒光的TM4細胞增多,以50 nM和100 nM最為明顯,選定50 nM作為siRNA的有效轉(zhuǎn)染濃度。三種si-uPA轉(zhuǎn)染TM4細胞后,uPA表達量均較空白對照組明顯下降(P0.05),以si-uPA1作用最為明顯,而試劑對照組uPA mRNA表達無明顯改變。用si-uPAl轉(zhuǎn)染TM4細胞進行時效量效實驗,結(jié)果顯示,轉(zhuǎn)染24 h后,轉(zhuǎn)染組細胞uPA mRNA的表達均較對照組顯著降低,其中100 nM組抑制效果最為明顯,抑制率達到70%,抑制效果強于30 nM組和50 nM組(P0.05)；隨轉(zhuǎn)染時間的延長,uPA mRNA表達持續(xù)降低,轉(zhuǎn)染72 h后,三組細胞uPA mRNA表達量分別為對照組的53.9%、35.3%和27.7%,差異具有統(tǒng)計學意義(P0.05)。 結(jié)論：本部分成功篩選出針對uPA mRNA靶序列的有效干擾序列si-uPA1,轉(zhuǎn)染TM4細胞后,在24 h即可抑制細胞uPA mRNA的表達,抑制效應(yīng)持續(xù)至72 h；同一濃度的si-uPA1在不同時間點內(nèi)的抑制效應(yīng)無顯著性差異；同一時間點內(nèi),抑制效應(yīng)隨轉(zhuǎn)染濃度的增加而增強,表現(xiàn)出良好的量效關(guān)系。 第二部分慢病毒介導(dǎo)的可誘導(dǎo)RNAi體系的建立 目的：在第一部分實驗基礎(chǔ)上構(gòu)建慢病毒介導(dǎo)的含有Tet調(diào)控元件的uPA干擾載體,進行病毒包裝和滴度測定后,感染TM4細胞,利用抗性篩選獲得TM4雙穩(wěn)轉(zhuǎn)細胞株,為后續(xù)Dox調(diào)控提供前提條件。 方法：根據(jù)第一部分實驗獲得的uPA-siRNA的有效干擾序列si-uPA1,設(shè)計合成uPA-shRNA oligo,退火成雙鏈,插入入門載體pENTR/H1/TO中,轉(zhuǎn)化感受態(tài)細胞,獲取入門克隆。利用Gateway技術(shù)的LR重組反應(yīng),將入門克隆中的Hl/TO-RNA干擾序列轉(zhuǎn)入慢病毒表達載體pLenti4-BLOCK-iT-DEST中,轉(zhuǎn)化感受態(tài)細胞,提取質(zhì)粒,測序驗證,構(gòu)建針對uPA的慢病毒干擾載體pLenti4-sh。將慢病毒表達載體pLenti4-sh和含有四環(huán)素抑制子(tetracycline repressor, TetR)的慢病毒載體pLenti6/TR分別轉(zhuǎn)染293T細胞,包裝病毒,收集病毒原液,超速離心濃縮,運用qRT-PCR技術(shù)測定病毒滴度。用不同濃度的抗生素以及帶EGFP的慢病毒原液(Lentil-EGFP)感染TM4細胞,選擇抗生素的最佳篩選濃度和病毒感染的感染復(fù)數(shù)(multiplicity of infection, MOI)值。在此基礎(chǔ)上,將pLenti6/TR慢病毒液感染TM4細胞,用Blasticidin篩選陽性細胞,qRT-PCR檢測TetR的表達,建立TetR穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR;將pLenti4-sh慢病毒液感染TM4-Lenti6/TR,用Blasticidin和Zeocin共同篩選陽性細胞,qRT-PCR檢測TetR和抗性基因的表達,建立雙穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR Lenti4-sh. 結(jié)果：針對uPA靶序列構(gòu)建的慢病毒干擾載體為pLenti4-sh,經(jīng)測序驗證,序列比對完全正確。慢病毒包裝后,qRT-PCR測定Lenti6/TR病毒的物理滴度為9.8×109vp/ ml, Lenti4-sh病毒的物理滴度為1.72×1010vp/ml。不同濃度抗生素作用TM4細胞后,根據(jù)細胞生長狀況觀察,選擇Blasticidin最佳篩選濃度為1μg/ml, Zeocin的最佳篩選濃度為600μg/ml.隨著感染TM4細胞的Lentil-EGFP MOI值的增大,表達EGFP的TM4細胞增多,熒光亮度增強。因而將慢病毒感染TM4細胞的MOI設(shè)立為100。根據(jù)2-ΔΔCt的方法相對定量,TM4-Lenti6/TR細胞中TetR基因的表達量為正常TM4細胞的27301.01倍,而TM4-Lenti6/TR Lenti4-sh細胞中TetR基因和抗性基因表達量分別正常TM4細胞的406623.29倍和51212760.84倍。 結(jié)論：成功構(gòu)建了慢病毒介導(dǎo)的含有Tet調(diào)控元件的uPA干擾載體。經(jīng)TM4細胞感染和抗性篩選后,建立了TetR穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR和雙穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR Lenti4-sh. 第三部分可誘導(dǎo)的RNAi調(diào)控uPA表達的實驗研究 目的：利用第二部分實驗中獲得的穩(wěn)轉(zhuǎn)細胞株和攜帶uPA干擾載體的慢病毒液,構(gòu)建體外細胞培養(yǎng)體系和小鼠在體動物模型,通過Dox給藥,觀察雙穩(wěn)轉(zhuǎn)細胞株和小鼠睪丸組織uPA mRNA和蛋白表達情況以及uPA酶活性的改變,評估Dox對uPA的誘導(dǎo)效應(yīng)。 方法：通過MTT實驗檢測不同濃度Dox對TM4細胞的毒性,篩選Dox作用安全劑量。在此基礎(chǔ)上,將第二部分獲得的雙穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR Lenti4-sh進行體外培養(yǎng),以TetR穩(wěn)轉(zhuǎn)細胞株TM4-Lenti6/TR和TM4細胞作為對照,通過Dox誘導(dǎo),在24 h、48 h、72 h觀察各組細胞uPA mRNA和蛋白表達的抑制情況以及細胞培養(yǎng)液中uPA酶活性的改變,評估Dox的體外誘導(dǎo)效應(yīng)。在Dox作用72 h后,移除Dox,觀察移除24 h、48 h、72 h后uPA mRNA和蛋白表達的恢復(fù)情況。將慢病毒液Lenti4-sh和Lenti6/TR經(jīng)皮睪丸注射,構(gòu)建小鼠實驗?zāi)Ｐ?以注射Lenti6/TR慢病毒液和生理鹽水的小鼠作為對照,通過Dox灌胃進行體內(nèi)誘導(dǎo),在Dox作用24 h、48 h、72 h和1周后觀察各組小鼠睪丸組織uPA mRNA和蛋白的表達情況以及睪丸組織勻漿液uPA酶活性變化。 結(jié)果：MTT實驗篩選得出Dox作用于TM4細胞的安全濃度在5μg/ml以下。以1μg/ml的Dox對TM4-TetRshuPA、TM4-TetR和TM4細胞進行體外誘導(dǎo),結(jié)果顯示,后兩組細胞uPA表達在誘導(dǎo)組和非誘導(dǎo)組間無明顯差異,而TM4-TetRshuPA雙穩(wěn)轉(zhuǎn)細胞組的uPA mRNA在Dox誘導(dǎo)24 h后即較非誘導(dǎo)組有明顯下降,隨誘導(dǎo)時間的延長,抑制作用更為明顯,作用72 h時,誘導(dǎo)組表達量僅為非誘導(dǎo)組的35%。uPA蛋白表達變化較mRNA出現(xiàn)略晚,在Dox誘導(dǎo)72 h后,蛋白表達較非誘導(dǎo)組顯著降低。S-2251發(fā)色底物法檢測結(jié)果示,雙穩(wěn)轉(zhuǎn)細胞誘導(dǎo)組uPA酶活性在72 h時明顯降低,為非誘導(dǎo)組的65.1%。隨著Dox從細胞培養(yǎng)液的移除,uPA表達逐漸恢復(fù),移除48 h后,uPAmRNA和蛋白表達水平與非誘導(dǎo)組無顯著性差異。 雄性小鼠經(jīng)皮睪丸內(nèi)注射慢病毒載體后,以0.75 mg/ml的Dox進行誘導(dǎo),結(jié)果表明,TetRshuPA小鼠在Dox作用24 h后,uPA mRNA水平較非誘導(dǎo)組以及對照組明顯降低,隨Dox作用時間的延長,uPA mRNA的表達逐漸減少,Dox作用1周后,抑制作用最為明顯,uPA mRNA表達水平與誘導(dǎo)24 h比較差異具有統(tǒng)計學意義。uPA蛋白與mRNA表達情況略有不同,在Dox作用的72 h內(nèi),TetRshuPA小鼠睪丸uPA蛋白與非誘導(dǎo)組表達水平相接近,而在Dox作用1周后,uPA蛋白表達水平在誘導(dǎo)組出現(xiàn)了明顯下降,與非誘導(dǎo)組有顯著差異。 結(jié)論：Dox對細胞培養(yǎng)體系和小鼠睪丸組織內(nèi)的uPA表達均有調(diào)控作用,其抑制uPA表達在體外體系可維持至72 h,移除Dox后,uPA表達水平在48 h內(nèi)逐漸恢復(fù)。小鼠在體模型中,Dox誘導(dǎo)可以下調(diào)睪丸組織中uPA表達水平以及勻漿液uPA酶活性,抑制作用至誘導(dǎo)1周最為明顯。 第四部分調(diào)控uPA表達影響雄性小鼠生育力的實驗研究 目的：通過第三部分動物體內(nèi)誘導(dǎo)實驗,觀察睪丸uPA表達受到可誘導(dǎo)的RNAi調(diào)控后,雄性小鼠生育力、精子功能的改變,分析uPA影響雄性小鼠生育力的可能機制。 方法：慢病毒液經(jīng)皮睪丸注射構(gòu)建雄性小鼠動物模型,Dox灌胃1周后行交配實驗,觀察雄鼠交配率、交配雌鼠妊娠率、平均胚胎數(shù)和黃體數(shù)。解剖雄鼠,測定附睪精子活動百分率和存活率,低滲腫脹實驗觀察精子膜功能。小鼠睪丸附睪取材,光鏡和透射電鏡觀察組織病理和超微結(jié)構(gòu)改變,并觀察動物生殖系統(tǒng)外其他組織器官病理變化。分別在Dox停藥2周、4周和8周再次與雌鼠交配,觀察雄鼠生育力和精子功能的恢復(fù)情況。 結(jié)果：Dox給藥1周后各組小鼠交配率無明顯差異。TetRshuPA小鼠誘導(dǎo)組雌鼠妊娠率和平均胎數(shù)較非誘導(dǎo)組和空白對照組顯著降低,差異具有統(tǒng)計學意義(P0.05),平均黃體數(shù)無顯著性差異。停藥2周后,TetRshuPA誘導(dǎo)組雌鼠妊娠率和平均胎數(shù)明顯增加,至停藥8周時,雌鼠妊娠率和平均胎數(shù)與非誘導(dǎo)組和空白對照組水平相當。 Dox誘導(dǎo)后,各組小鼠的精子存活率和精子低滲腫脹率均無明顯差異。TetRshuPA小鼠活動精子百分率較非誘導(dǎo)組和空白對照組明顯下降(P0.05)。隨Dox停藥時間的延長,TetRshuPA誘導(dǎo)組小鼠精子活力逐漸恢復(fù),至停藥8周時,誘導(dǎo)組小鼠精子活力恢復(fù)至空白對照組水平。 Dox用藥后,各組小鼠睪丸附睪組織結(jié)構(gòu)和超微結(jié)構(gòu)未見明顯病理改變。實驗組小鼠其他組織器官未見病理性結(jié)構(gòu)損傷。 結(jié)論：Dox體內(nèi)誘導(dǎo)睪丸uPA表達下調(diào)后不影響雄鼠的交配率及雌鼠黃體數(shù),交配雌鼠的妊娠率和平均胎仔數(shù)下降。uPA的下調(diào)不影響精子存活率和精子膜功能,但可導(dǎo)致雄鼠精子活力降低。雄鼠生育力和精子活力在Dox停藥8周后可完全恢復(fù)。uPA下調(diào)對雄鼠睪丸、附睪病理結(jié)構(gòu)和超微結(jié)構(gòu)無明顯病理影響,雄鼠的心、肝、脾、肺、腎等主要臟器形態(tài)未見明顯損傷性改變。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R346

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