本文關(guān)鍵詞： 干細(xì)胞白血病基因發(fā) 慢病毒 Cajal樣間質(zhì)細(xì)胞 糖尿病膀胱病　出處：《石河子大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:1.觀察含綠色熒光蛋白(GFP)基因的干細(xì)胞白血病基因(SCL)重組慢病轉(zhuǎn)染體外高糖環(huán)境下膀胱Cajal樣間質(zhì)細(xì)胞的形態(tài)學(xué)變化。2.觀察經(jīng)尿道灌注含綠色熒光蛋白(GFP)基因的SCL基因重組慢病毒轉(zhuǎn)染豚鼠DCP(diabetic cystopathy,DCP)膀胱對Cajal樣間質(zhì)細(xì)胞的形態(tài)學(xué)影響。方法:1.構(gòu)建SCL基因重組慢病毒且標(biāo)定滴度;2.體外分離培養(yǎng)豚鼠膀胱ICC(interstitial cells of Cajal,ICC),分別用5mmol/L,15mmol/l和25mmol/l葡萄糖濃度處理24小時,然后每個濃度下分為空白對照組(加1%PBS),空慢病毒對照組(加空慢病毒),慢病毒轉(zhuǎn)染組(加攜帶人SCL基因慢病毒),轉(zhuǎn)染2d、3d、5d時,用激光共聚焦顯微鏡觀測各組ICC的狀態(tài)和數(shù)量變化及GFP表達(dá)。3.構(gòu)建DCP豚鼠模型;觀察經(jīng)尿道灌注SCL基因重組慢病毒對豚鼠DCP膀胱組織中ICC數(shù)量、分布及超微結(jié)構(gòu)的影響;結(jié)果:1.病毒滴度測定是5×108TU/ml;2.顯微鏡下可見典型的膀胱ICC形態(tài)及高葡萄糖濃度(15 mmol/L和25 mmol/L)受損的細(xì)胞形態(tài),免疫熒光法可見特異性c-kit受體成功表達(dá);慢病毒轉(zhuǎn)染成功導(dǎo)入SCL基因至ICC中;在15 mmol/L葡萄糖濃度下,SCL基因的導(dǎo)入可陸續(xù)恢復(fù)形態(tài)損傷的ICC。在25 mmol/L葡萄糖濃度下,能夠改善卻沒能恢復(fù)形態(tài)損傷的ICC,此外,SCL基因未使ICC數(shù)量得到恢復(fù)或增殖。3.糖尿病豚鼠誘導(dǎo)成功率為47.7%;誘導(dǎo)DCP豚鼠27只;激光共聚焦顯微鏡下觀察發(fā)現(xiàn):實(shí)驗(yàn)組中隨7d、14d、28d時間的延長,ICC數(shù)量逐漸增多(P0.05),分布逐漸密集,綠色熒光逐漸減弱�？瞻讓φ战M和陽性對照組中隨7d、14d、28d時間的延長,ICC數(shù)量逐漸減少(P0.05),分布稀疏,因無GFP表達(dá),未見綠色熒光;透射電鏡下觀察發(fā)現(xiàn):實(shí)驗(yàn)組中隨7d、14d、28d時間的延長,ICC的細(xì)胞器數(shù)量增多、形態(tài)趨于正常、細(xì)胞內(nèi)空泡減少、胞周突起延長變多,受損的ICC的超微結(jié)構(gòu)在不斷的恢復(fù)之中�？瞻讓φ战M和陽性對照組中隨7d、14d、28d時間的延長,ICC的胞器數(shù)量減少、形態(tài)不規(guī)則、胞內(nèi)空泡樣變增多、胞周突起稀少,ICC的超微結(jié)構(gòu)未見改善。結(jié)論:1.SCL基因重組慢病毒載體轉(zhuǎn)染體外高糖環(huán)境下培養(yǎng)的ICC,可將SCL基因?qū)隝CC中,能夠恢復(fù)或改善受損的ICC形態(tài)。2.經(jīng)尿道灌注SCL基因重組慢病毒轉(zhuǎn)染豚鼠DCP膀胱后,對ICC形態(tài)和數(shù)量的恢復(fù)有一定收效,對DCP達(dá)到一定程度的治療效果。能進(jìn)一步為使用SCL基因重組慢病毒對DCP的基因治療提供思路。
[Abstract]:Objective 1. To observe the morphological changes of bladder Cajal like interstitial cells transfected with recombinant stem cell leukemia gene (SCL) containing green fluorescent protein (GFP) gene in vitro. 2. To observe the transurethral perfusion of GFP gene containing green fluorescent protein (GFP). The effects of recombinant lentivirus of SCL gene on the morphology of Cajal-like interstitial cells in guinea pig DCP(diabetic cystopathyte) bladder. Methods 1. Construction of recombinant lentivirus of SCL gene and calibration titer 2. Isolation and culture of ICC(interstitial cells of Cajalalus in vitro, 5 mmol / L / 15 mmol / L and 25 mmol / 1, respectively. Glucose concentration was treated for 24 hours. Then each concentration was divided into two groups: blank control group (add 1 PBSX), empty lentivirus control group (plus empty lentivirus), lentivirus transfection group (plus lentivirus carrying human SCL gene), transfection of human SCL gene lentivirus for 3 days or 5 days after transfection, The changes of ICC and the expression of GFP in each group were observed by confocal laser microscope. The DCP guinea pig model was established, and the effects of transurethral perfusion of SCL gene recombinant lentivirus on the quantity, distribution and ultrastructure of ICC in DCP bladder were observed. Results 1. The virus titer was 5 脳 108 TU / ml 2.The typical ICC morphology of bladder and the damaged cells with high glucose concentration of 15 mmol/L and 25 mmol / L were observed under microscope, and the specific c-kit receptor was successfully expressed by immunofluorescence. SCL gene was successfully transfected into ICC by lentivirus transfection, and the morphological damage could be recovered at 15 mmol/L glucose concentration. The number of ICC was not recovered or proliferated by ICC gene. The successful rate of induction was 47.7% in diabetic guinea pigs and 27 in DCP guinea pigs. The results of laser confocal microscopy showed that the number of ICC increased gradually with the extension of 14 d and 28 d in the experimental group, and the distribution of ICC increased gradually. The number of GFP decreased gradually in the blank control group and the positive control group with the extension of 14d ~ 28d, and the distribution was sparse. There was no GFP expression and no green fluorescence was found in the control group. The results of transmission electron microscope showed that the number of organelles increased, the morphology of ICC tended to be normal, the vacuoles decreased, and the pericellular processes became longer in the experimental group with the extension of 14d ~ 28d. The ultrastructure of the damaged ICC was recovering continuously. The number of organelles in the control group and the positive control group decreased with the prolongation of 14d ~ 28d, and the morphology was irregular, and the vacuolation became more and more in the control group and the positive control group. Conclusion: 1. The recombinant lentivirus vector of SCL gene can be transfected into ICC in high glucose environment, and the SCL gene can be transferred into ICC. After transfection of SCL gene recombinant lentivirus into guinea-pig DCP bladder, the morphology and quantity of ICC recovered to some extent. The therapeutic effect on DCP can be achieved to a certain extent, which can further provide ideas for the gene therapy of DCP using SCL gene recombinant lentivirus.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R694

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