【摘要】：第一部分ECSOD基因表達(dá)載體的構(gòu)建并轉(zhuǎn)染至骨髓間充質(zhì)干細(xì)胞的實驗研究 實驗?zāi)康模簶?gòu)建胞外超氧化物歧化酶(extracellular supero-xide dismutase, ECSOD)基因表達(dá)載體,經(jīng)慢病毒包裝后轉(zhuǎn)染至成年Sprague-Dawley(SD)雄性大鼠的骨髓間充質(zhì)干細(xì)胞(bone mar-row mesenchymal stem cell, MSCs),觀察和檢測ECSOD基因的轉(zhuǎn)染效率及轉(zhuǎn)染后對細(xì)胞活力的影響與ECSOD蛋白表達(dá)情況。 實驗方法：利用基因工程技術(shù)從MSCs中提取總的RNA,經(jīng)PCR擴(kuò)增、載體雙酶切后,獲得ECSOD基因片段,將其基因片段連接到GV230-EGFP載體構(gòu)建GV230-EGFP-ECSOD表達(dá)載體,經(jīng)菌落PCR及測序,鑒定載體構(gòu)建是否成功,采用慢病毒包裝后將GV230-EGFP-ECSOD導(dǎo)入MSCs中,熒光顯微鏡檢測轉(zhuǎn)染效率,實時熒光定量多聚酶鏈反應(yīng)(Quantitati-ve Polymerase Chain Reaction, QPCR)對轉(zhuǎn)染后對MSCs中mRNA定量分析,MTT及EDU檢測轉(zhuǎn)染后ECSOD基因?qū)?xì)胞活力與增殖影響,Western Blot檢測ECSOD基因表達(dá)情況。 實驗結(jié)果：提取RNA產(chǎn)物經(jīng)PCR擴(kuò)增、雙酶切成功將目的基因連接至真核表達(dá)載體GV230-EGFP中,菌落PCR獲得目的基因大小和陽性對照一致,菌落PCR陽性轉(zhuǎn)化子經(jīng)測序分析與目的基因序列完全一致；慢病毒包裝的GV230-EGFP-ECSOD成功轉(zhuǎn)染至MSCs,培養(yǎng)48h后在熒光顯微鏡下觀察并計算得MSCs轉(zhuǎn)染率為(75±3.6)%,進(jìn)一步采用QPCR定量測定轉(zhuǎn)染后mRNA含量明顯高于對照組。EDU與MTT檢測發(fā)現(xiàn)ECSOD基因轉(zhuǎn)染MSCs后對細(xì)胞活力無影響。Western Blot檢測證實ECSOD基因轉(zhuǎn)染MSCs后,MSCs中ECSOD蛋白呈陽性表達(dá)。 實驗結(jié)論：成功構(gòu)建GV230-EGFP-ECSOD真核表達(dá)載體,通過慢病毒包裝轉(zhuǎn)染技術(shù)將ECSOD基因轉(zhuǎn)染到MSCs中且表達(dá)ECSOD蛋白,進(jìn)而為下一步研究ECSOD基因轉(zhuǎn)染MSCs治療腦梗塞動物模型奠定實驗基礎(chǔ)。 第二部分ECSOD基因轉(zhuǎn)染MSCs移植治療缺血性腦卒中大鼠神經(jīng)保護(hù)作用的實驗研究 實驗?zāi)康模河^察ECSOD基因轉(zhuǎn)染的MSCs移植治療缺血性腦卒中大鼠的神經(jīng)保護(hù)作用。 實驗方法：將80只Sprague DaWley (SD)成年雄性大鼠,隨機分為兩組6h組(n=40)、12h組(n=40),分別將6h、12h組分為四個亞組：Model組(n=10)、PBS組(n=10)、MSCs組(n=10)、ECSOD-MSCs組(n=10)；根據(jù)改良Zea Longa線栓法制作左側(cè)大腦中動脈缺血模型,分別在造模成功后6h、12h腦缺血/再灌注,同時經(jīng)顱立體定位對后三組腦缺血大鼠分別注射10μLPBS液、MSCs液、ECSOD-MSCs液,Model組不做處理。腦缺血/再灌注后Id、3d、7d、2w、4w進(jìn)行改良神經(jīng)功能評分(Modified Neurological Severity Score, mNSS)評估預(yù)后,缺血/再灌注后1天、28天分別行MRI檢查測定大鼠腦梗塞體積變化情況。4周后取腦組織采用免疫組織化學(xué)方法測定腦組織梗塞周圍ECSOD、Bax、Bcl-2的表達(dá)情況。 實驗結(jié)果：移植后2w、4w, ECSOD-MSCs組和MSCs組的mNSS評分較PBS組、Model組低(P0.05),其中ECSOD-MSCs組mNSS評分更低。移植后腦梗塞體積變化的相對值比較ECSOD-MSCs組、MSCs組明顯大于PBS組、Model組,差異有統(tǒng)計學(xué)意義(P0.05),其中ECSOD-MSCs組更大。在腦梗塞移植后28d腦梗塞及周圍ECSOD、Bax、Bcl-2的表達(dá)情況：ECSOD-MSCs組較MSCs組、PBS組、Model組ECSOD表達(dá)高,差異有統(tǒng)計學(xué)差異(P0.05); ECSOD-MSCs組、MSCs組較PBS組、Model組Bax陽性細(xì)胞數(shù)明顯少(P0.05), ECSOD組更少；而bcl-2陽性細(xì)胞數(shù)ECSOD-MSCs組、MSCs組高于Model組、PBS組(P0.05),其中ECSOD組更高。 實驗結(jié)論：ECSOD基因轉(zhuǎn)染MSCs細(xì)胞能預(yù)防腦缺血后再灌注損傷,進(jìn)一步改善大鼠神經(jīng)功能缺損,挽救梗死邊緣的腦組織。
[Abstract]:Part one: Construction and transfection of ECSOD gene expression vector into bone marrow mesenchymal stem cells
OBJECTIVE: To construct an extracellular supero-oxide dismutase (ECSOD) gene expression vector and transfect it into adult Sprague-Dawley (SD) male rat bone Mar row mesenchymal stem cells (MSCs) after packaging with lentivirus. The effect on cell viability and ECSOD protein expression.
METHODS: Total RNA was extracted from MSCs by genetic engineering technology, amplified by PCR, and digested by double enzyme. The ECSOD gene fragment was obtained. The gene fragment was linked to GV230-EGFP vector to construct GV230-EGFP-ECSOD expression vector. The vector was successfully constructed by colony PCR and sequencing. The vector was packaged with lentivirus and then guided by GV230-EGFP-ECSOD. In MSCs, the transfection efficiency was examined by fluorescence microscope, the mRNA in MSCs was quantitatively analyzed by real-time fluorescence quantitative polymerase chain reaction (QPCR), the effect of ECSOD gene on cell viability and proliferation was detected by MTT and EDU, and the expression of ECSOD gene was detected by Western Blot.
The results showed that the RNA product was amplified by PCR, and the target gene was successfully linked to the eukaryotic expression vector GV230-EGFP by double digestion. The size of the target gene obtained by colony PCR was identical with that of the positive control. The positive transformant of colony PCR was completely identical with the target gene sequence by sequencing analysis. The lentivirus-packaged GV230-EGFP-ECSOD was successfully transfected into MSCs. After 48 hours of culture, the transfection rate of MSCs was (75 Da.
Conclusion: Eukaryotic expression vector of GV230-EGFP-ECSOD was successfully constructed, and ECSOD gene was transfected into MSCs by lentiviral packaging transfection technology to express ECSOD protein. This will lay an experimental foundation for further study of ECSOD gene transfected MSCs to treat cerebral infarction animal model.
Part 2 Experimental study on neuroprotective effect of ECSOD gene transfected MSCs transplantation on ischemic stroke rats
Objective: To observe the neuroprotective effect of MSCs transfected with ECSOD gene on ischemic stroke rats.
Methods: 80 Sprague DaWley (SD) adult male rats were randomly divided into two groups: 6 h group (n = 40) and 12 h group (n = 40). The rats were divided into four subgroups: Model group (n = 10), PBS group (n = 10), MSCs group (n = 10), ECSOD-MSCs group (n = 10), and left middle cerebral artery ischemia model was established by modified Zea Longa occlusion method. Six hours and twelve hours after cerebral ischemia/reperfusion, and three groups of rats were injected with 10 mu LPBS, MSCs, ECSOD-MSCs respectively, without treatment in the Model group. Modified Neurological Severity Score (mNSS) was used to evaluate the prognosis of the rats at the 1st and 28th days after cerebral ischemia/reperfusion. The changes of infarct volume were detected by MRI. The expression of ECSOD, Bax and Bcl-2 were detected by immunohistochemistry in the brain tissues around infarction 4 weeks later.
The results showed that the mNSS scores of ECSOD-MSCs and MSCs groups were lower than those of PBS and Model groups (P 0.05), and the mNSS scores of ECSOD-MSCs group were lower than those of PBS group and Model group (P 0.05). The expression of ECSOD, Bax and Bcl-2 was significantly higher in ECSOD-MSCs group than in MSCs group, PBS group and Model group at 28 days after transplantation (P 0.05); the number of Bax-positive cells in ECSOD-MSCs group was significantly lower than that in PBS group, MSCs group, Model group and ECSOD-MSCs group (P 0.05), while the number of bcl-2-positive cells in ECSOD-MSCs group was higher than that in Model group. Group PBS and group P0.05 (ECSOD) were higher.
CONCLUSION: ECSOD gene transfection into MSCs cells can prevent reperfusion injury after cerebral ischemia, further improve neurological deficits in rats, and save the brain tissue at the edge of infarction.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳朝;郭建文;黃燕;;骨髓間充質(zhì)干細(xì)胞移植治療缺血性腦損傷的研究現(xiàn)狀[J];中國醫(yī)藥導(dǎo)刊;2009年10期

2 陳大帥;周思遠(yuǎn);唐宏智;李瑛;;針刺調(diào)節(jié)核轉(zhuǎn)錄因子-κB信號通路的實驗研究進(jìn)展[J];中華中醫(yī)藥雜志;2013年10期

3 龔志剛;丁世芳;李志剛;;炎癥和氧化應(yīng)激對內(nèi)皮祖細(xì)胞動員及其功能的影響[J];中國組織化學(xué)與細(xì)胞化學(xué)雜志;2013年06期

4 林亨;陳兵;黃梓雄;莫偉;李小亭;朱鵬博;尹延慶;廖壯檳;;劃痕損傷對大鼠腦皮層星形膠質(zhì)細(xì)胞NF-êB表達(dá)及繼發(fā)性死亡的影響[J];廣東醫(yī)學(xué)院學(xué)報;2014年04期

5 尚全良;肖恩華;周啟昌;吳海軍;;同種異體骨髓單個核細(xì)胞肝內(nèi)移植治療急性肝損傷的MVD改變及VEGF的表達(dá)(英文)[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2009年08期

6 陳朝;黎奔;郭建文;;大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及CM-Dil標(biāo)記的腦內(nèi)示蹤[J];解放軍醫(yī)學(xué)雜志;2010年08期

7 孔衛(wèi)國;吳曉牧;;骨髓間充質(zhì)干細(xì)胞移植治療腦梗死的作用機制[J];中國腦血管病雜志;2010年04期

8 彭亞;宣井崗;陳榮華;朱旭成;楊伊林;;Solitaire AB支架取栓術(shù)治療急性大腦中動脈閉塞八例[J];中國腦血管病雜志;2011年07期

9 繆中榮;;急性腦梗死溶栓治療的新模式[J];中國腦血管病雜志;2011年02期

10 王旭;張志彬;賈芙蓉;楊宏;;自體骨髓間充質(zhì)干細(xì)胞介入移植治療腦梗死臨床研究[J];吉林醫(yī)學(xué);2014年02期

相關(guān)會議論文 前2條

1 鄒凱杰;高海s，

本文編號：2251293