【摘要】：研究目的1.研究熱休克蛋白90α功能性基因序列(以下稱“F-5”基因)的慢病毒載體轉(zhuǎn)染間充質(zhì)干細胞(Mesenchymal Stem Cells,MSC)后對間充質(zhì)干細胞生物活性的影響及“F-5”基因在MSC的表達情況;2.探討間充質(zhì)干細胞聯(lián)合轉(zhuǎn)染“F-5”基因移植治療大鼠皮瓣缺血再灌注損傷的臨床效果。研究方法1.將構(gòu)建好的“F-5”基因慢病毒載體體外轉(zhuǎn)染間充質(zhì)干細胞,確定最佳的病毒感染復數(shù),在鏡下觀察細胞形態(tài)、繪制細胞的生長曲線、測定細胞周期、劃痕實驗和MMT檢測轉(zhuǎn)染后的間充質(zhì)干細胞的生物學活性;熒光顯微鏡和細胞免疫熒光檢測“F-5”多肽在間充質(zhì)干細胞中的表達情況;Elisa分別檢測細胞上清液中F-5多肽在1d、3d、5d、7d和14d的分泌情況。2.設“F-5”基因慢病毒載體轉(zhuǎn)染后的間充質(zhì)干細胞為實驗組、空載慢病毒載體為對照組、1×PBS為空白組,分別注射移植于缺血再灌注損傷的大鼠腹部皮瓣術(shù)區(qū);術(shù)后每天觀察皮瓣的顏色、質(zhì)地、毛發(fā)生長、壞死范圍及針刺出血情況等,術(shù)后7 d比較各組皮瓣的成活率,并切取成活皮瓣遠端組織分別進行HE染色、免疫組織化學染色檢測血管內(nèi)皮生長因子(VEGF)的表達,并用TUNEL法檢測凋亡細胞,比較各組間的差異。結(jié)果:1.攜帶“F-5”基因的慢病毒載體轉(zhuǎn)染間充質(zhì)干細胞后,通過鏡下觀察的細胞形態(tài)、繪制細胞的生長曲線、測定細胞的周期、劃痕實驗和MMT檢測并未見明顯差異;通過細胞免疫熒光檢測發(fā)現(xiàn)在轉(zhuǎn)染后的間充質(zhì)干細胞中的細胞質(zhì)和細胞膜上均能夠檢測到“F-5”多肽的表達;通過Elisa檢測法發(fā)現(xiàn)實驗組從第3天開始細胞上清液中分泌的F-5-flag的量明顯比其他兩組分泌量多,第5天時可發(fā)現(xiàn)實驗組比其他兩組明顯高出10倍的量。2.實驗組皮瓣成活率為(92.38±8.67)%,而對照組為(81.57±7.38)%,空白組為(47.83+6.89)%。實驗組大鼠皮瓣VEGF的表達密度為(91.25±5.92)%,對照組的為(76.37±6.42)%和空白組的為(33.37±6.62)%;各組凋亡細胞陽性表達百分指數(shù)分別為:實驗組(21.4±5.3)%,對照組(23.3±3.6)%,空白組為(54.7±5.7)%。結(jié)論及意義:1.“F-5”基因慢病毒載體轉(zhuǎn)染間充質(zhì)干細胞后對細胞的生物活性無明顯影響,“F-5”基因能夠在MSC中成功表達并將F-5多肽分泌至細胞外,同時本實驗為“F-5”基因在組織修復等研究工作中提供了實驗基礎;2.間充質(zhì)干細胞聯(lián)合轉(zhuǎn)染“F-5”基因治療大鼠皮瓣缺血再灌注損傷的效果明顯優(yōu)于單純MSC移植的效果。
[Abstract]:Objective 1. Study on transfection of heat shock protein 90 偽 functional gene sequence (hereinafter referred to as "F-5" gene) lentivirus vector into mesenchymal stem cell (Mesenchymal Stem Cells, The effect of MSC on the biological activity of mesenchymal stem cells and the expression of "F-5" gene in MSC; 2. To investigate the clinical effect of mesenchymal stem cells (MSCs) combined with transfection of F-5 gene in the treatment of ischemia reperfusion injury of skin flap in rats. Method 1. The "F-5" gene lentivirus vector was transfected into mesenchymal stem cells in vitro, and the optimal number of virus infection was determined. The morphology of cells was observed under microscope, the growth curve of cells was drawn, and the cell cycle was measured. The biological activity of transfected mesenchymal stem cells was detected by scratch test and MMT. The expression of "F-5" polypeptide in mesenchymal stem cells was detected by fluorescence microscope and immunofluorescence, and the secretion of F-5 polypeptide in supernatant was detected by Elisa for 7 days and 14 days, respectively. Mesenchymal stem cells transfected with "F-5" gene lentivirus vector were used as experimental group, non-loaded lentivirus vector as control group and 1 脳 PBS as blank group. The color, texture, hair growth, necrotic range and acupuncture bleeding were observed every day after operation. The survival rate of each flap was compared 7 days after operation, and the distal tissue of the surviving flap was stained with HE. The expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical staining, and the apoptotic cells were detected by TUNEL method. Results: 1. After transfection of lentivirus vector carrying "F-5" gene into mesenchymal stem cells, cell morphology was observed under microscope, cell growth curve was drawn and cell cycle was measured. There was no significant difference between scratch test and MMT test. The expression of "F-5" polypeptide was detected in the cytoplasm and cell membrane of the transfected mesenchymal stem cells by immunofluorescence assay. The amount of F-5-flag secreted in the supernatant of the experimental group from the third day was obviously higher than that in the other two groups by Elisa assay. On the fifth day, the amount of F-5-flag secreted in the experimental group was 10 times higher than that in the other two groups. The survival rate of flap was (92.38 鹵8.67)% in the experimental group, (81.57 鹵7.38)% in the control group and (47.83.6.89)% in the blank group. The expression density of VEGF was (91.25 鹵5.92)% in the experimental group, (76.37 鹵6.42)% in the control group and (33.37 鹵6.62)% in the blank group. The positive expression index of apoptotic cells was (21.4 鹵5.3)% in the experimental group, (23.3 鹵3.6)% in the control group and (54.7 鹵5.7)% in the blank group. Conclusion: 1. After transfection of "F-5" gene lentivirus vector into mesenchymal stem cells, the biological activity of the cells was not significantly affected. "F-5" gene could be successfully expressed in MSC and secreted into the cells. 2. At the same time, this experiment provides the experimental basis for the study of "F-5" gene in tissue repair. 2. The effect of mesenchymal stem cells combined with transfection of "F-5" gene on ischemic reperfusion injury of rat flap was better than that of MSC transplantation alone.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R622

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