【摘要】：目的 探討肌源性干細(xì)胞(Muscle derived stem cells,MDSCs)在修復(fù)大鼠脊髓損傷過程中Notch1基因的表達(dá)差異。 方法 1、采用機(jī)械-混合酶消化法分離培養(yǎng)大鼠MDSCs,結(jié)蛋白(Desmin)細(xì)胞免疫化學(xué)鑒定其為肌源性干細(xì)胞。NFG誘導(dǎo)MDSCs并采用免疫組織化學(xué)法檢測誘導(dǎo)后的細(xì)胞表達(dá)NSE特異性標(biāo)志物。 2、根據(jù)實(shí)驗(yàn)要求,選取120只大鼠樣本,動物按隨機(jī)數(shù)目表法分為:A組:NGF誘導(dǎo)后且BrdU標(biāo)記的MDSCs組(n=30);B組:未誘導(dǎo)且BrdU標(biāo)記的MDSCs組(n=30);C組:對照組(n=30)及D組:空白對照組(n=30)。A、B、C組按取材時間不同分為1周、4周、8周。 3、手術(shù)方法:制作脊髓半橫斷模型。脊髓損傷后使用微量注射器分別向A組注射進(jìn)行NGF誘導(dǎo)分化后且BrdU標(biāo)記的MDSCs培養(yǎng)液,向B組注射等量未經(jīng)誘導(dǎo)分化但經(jīng)BrdU標(biāo)記的MDSCs培養(yǎng)液,C組注入相同劑量的PBS液。 4、各組大鼠在存活期間按不同時間點(diǎn)測定各組大鼠后肢運(yùn)動功能(BBB運(yùn)動功能評分及斜板實(shí)驗(yàn))。 5、免疫組化染色檢測觀察MDSCs在大鼠體內(nèi)的存活、遷移及分化情況。 6、RT-PCR和Western技術(shù)檢測肌源性干細(xì)胞植入后Notch通路中關(guān)鍵基因的表達(dá)情況。 結(jié)果 1、經(jīng)過機(jī)械-混合酶消化和差速貼壁后,成功培養(yǎng)出高純度、高活性的MDSCs,細(xì)胞免疫化學(xué)結(jié)果為Desmin(+)(陽性率80%)。 2、誘導(dǎo)過程中,細(xì)胞生長狀態(tài)良好,形態(tài)呈漸進(jìn)性變化,未見神經(jīng)球形成;誘導(dǎo)組7d后神經(jīng)樣細(xì)胞的胞體和突起NSE染色呈強(qiáng)陽性(37.29±1.59)%,而未誘導(dǎo)組的細(xì)胞陽性細(xì)胞率極低(4.26±0.34)%,兩組之間差異明顯(P0.01);誘導(dǎo)組的MDSCs樣品中檢測到NSE表達(dá)水平高于未誘導(dǎo)組(P0.01)。 3、行為觀察結(jié)果:傷后1周時A組(誘導(dǎo)組)的評分略高于B組(未誘導(dǎo)組)但均高于C組(對照組),隨著時間的延長,各組差異逐漸明顯,A組的評分明顯高于B組、明顯優(yōu)于未加干預(yù)的C組。結(jié)果顯示,經(jīng)NGF誘導(dǎo)肌源性干細(xì)胞移植治療脊髓損傷的療效明顯優(yōu)于單獨(dú)應(yīng)用肌源性干細(xì)胞移植的方法。 4、組織學(xué)結(jié)果:觀察BrdU標(biāo)記后移植的MDSCs在大鼠體內(nèi)的存活、遷移及分化情況。A組損傷修復(fù)尤其明顯,細(xì)胞數(shù)量多于B組。結(jié)果顯示,經(jīng)NGF誘導(dǎo)后的MDSCs在大鼠體內(nèi)修復(fù)脊髓損傷效果優(yōu)于單獨(dú)應(yīng)用的MDSCs。 5、RT-PCR及Western檢測結(jié)果顯示:在大鼠脊髓損傷過程中Notch信號通路中的Notch1節(jié)點(diǎn)基因mRNA及Notch1蛋白均呈現(xiàn)低表達(dá),誘導(dǎo)組和未誘導(dǎo)組有顯著差異(P0.05)。 結(jié)論 1、本實(shí)驗(yàn)采用機(jī)械-混合酶消化法進(jìn)行體外培養(yǎng)MDSCs的方法原代培養(yǎng)獲得了高純度、高活性的MDSCs,Desmin細(xì)胞免疫化學(xué)方法鑒定證明。 2、本實(shí)驗(yàn)采用NGF對MDSCs進(jìn)行體外誘導(dǎo)分化成神經(jīng)樣細(xì)胞植入大鼠脊髓損傷處進(jìn)行治療,取得一定效果,這對MDSCs做為種子細(xì)胞修復(fù)脊髓損傷有重要意義。 3、在大鼠脊髓損傷過程中Notch信號通路中的Notch1mRNA及Notch1蛋白均呈現(xiàn)低表達(dá),Notch信號分子低表達(dá)可促進(jìn)MDSCs分化為神經(jīng)樣細(xì)胞。
[Abstract]:Objective to investigate the difference of expression of Notch1 gene in the repair of spinal cord injury (sci) by myogenic stem cells (Muscle derived stem cells). Methods 1. Rat MDSCs were isolated and cultured by mechanical-mixed enzyme digestion. The desmin (Desmin) cells were identified by immunocytochemistry as myogenic stem cells. NFG-induced MDSCs and the expression of induced MDSCs were detected by immunohistochemistry. NSE specific markers. 2, according to the experimental requirements, 120 rat samples were selected. Animals were randomly divided into two groups according to the random number table: group A, induced by BrdU and labeled with BrdU, group B, MDSCs group without induction and labeled with BrdU, group C, group C: control group (nong30) and group D, group D: blank control group (nong30). Group A, B, C were divided into 1 week, 4 weeks and 8 weeks, according to the time of sampling, they were divided into 1 week, 4 weeks and 8 weeks, according to the time of sampling, they were divided into two groups: 1 week, 4 weeks and 8 weeks, respectively. 3. Operative method: the model of spinal cord hemitransection was made. After spinal cord injury, microsyringes were injected into group A with BrdU labeled MDSCs medium after NGF induction. The same dose of PBS solution was injected into group B with the same dose of PBS in group C, which was not induced to differentiate but was labeled by BrdU. 4. The hind limb motor function (BBB) was measured at different time points during the survival of rats in each group. (5) Immunohistochemical staining was used to observe the survival of MDSCs in rats. The expression of key genes in the Notch pathway after myogenic stem cell implantation was detected by RT-PCR and Western. Results 1. After mechanically mixed enzyme digestion and differential adherence, high purity and high activity MDSCs were successfully cultured. The positive rate of Desmin () (was 80%. 2. During induction, the cells grew well. The morphology showed progressive changes, and no neurosphere formation was found. After 7 days, the NSE staining of neuronal cells and neurites was strongly positive in the induction group (37.29 鹵1.59), but the positive cell rate in the uninduced group was very low (4.26 鹵0.34), the difference between the two groups was significant (P0.01), and the NSE expression level in the MDSCs samples of the induced group was higher than that in the uninduced group (P0.01). The scores of group A (induction group) were slightly higher than group B (no induction group) but higher than group C (control group) at 1 week after injury, and the scores of group A were higher than those of group C (control group) at 1 week after injury (P0.01), and the scores of group A were higher than those of group C (control group). The scores of group A were significantly higher than that of group B, and the scores of group A were better than those of group C without intervention. The results showed that the effect of NGF induced myogenic stem cell transplantation on spinal cord injury was significantly better than that of myogenic stem cell transplantation alone. 4. Histological results: the survival of BrdU labeled MDSCs in rats was observed. Migration and differentiation were more obvious in group A than in group B. the number of cells in group A was more than that in group B. The results show that The effect of MDSCs induced by NGF on the repair of spinal cord injury in rats was better than that of MDSCs.5 RT-PCR and Western alone. The results showed that the expression of Notch1 node gene mRNA and Notch1 protein in Notch signaling pathway was low in the process of spinal cord injury in rats. There was significant difference between the induced group and the uninduced group (P0.05). Conclusion 1. The method of mechanical-mixed enzyme digestion was used to culture MDSCs in vitro. The identification of high activity MDSCS Desmin cell immunocytochemistry proved that. 2. NGF was used to treat rat spinal cord injury induced by MDSCs in vitro. This is of great significance for the repair of spinal cord injury by MDSCs as seed cells. 3. In the process of spinal cord injury in rats, the low expression of Notch1mRNA and Notch1 protein in Notch signaling pathway can promote the differentiation of MDSCs into neuron-like cells.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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相關(guān)期刊論文 前1條

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本文編號：2170596