發(fā)布時(shí)間：2018-05-29 11:57

  本文選題：慢病毒 + 膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子　； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：研究背景隨著現(xiàn)代化社會(huì)發(fā)展,工業(yè)用電及生活用電的使用日益頻繁,人類(lèi)在日常生產(chǎn)及生活中因意外接觸造成的電損傷常有發(fā)生。人體作為導(dǎo)電體,接觸電流后,因電流通過(guò)組織產(chǎn)生高熱而造成損傷,故臨床上也稱(chēng)為電燒傷。電流通過(guò)同一橫截面時(shí),電阻越大的部位,產(chǎn)熱越高,對(duì)人體造成損傷也越明顯,因此皮膚骨骼等部位損傷嚴(yán)重,神經(jīng)損傷較輕。同時(shí)根據(jù)電流傳導(dǎo)的方向最初接觸皮膚進(jìn)入人體的部位為電流入口,經(jīng)組織傳導(dǎo)流出體外的部位為電流出口。電流接觸或流出皮膚的瞬間均可能產(chǎn)生放電現(xiàn)象,對(duì)人體造成損傷,損傷程度甚至高于熱損傷,電流出口放電現(xiàn)象更明顯,因而組織損傷更嚴(yán)重。由于接觸放電造成損傷創(chuàng)面的修復(fù)目前臨床上手段多樣,效果良好。然而電流通過(guò)時(shí)產(chǎn)熱或引起組織其他病理改變而造成的血管栓塞、肌肉壞死、神經(jīng)變性等問(wèn)題目前還無(wú)法得到有效的解決。據(jù)統(tǒng)計(jì),電損傷多緣于四肢接觸,周?chē)窠?jīng)損傷多發(fā),目前臨床上缺乏有效的對(duì)神經(jīng)損傷的修復(fù)手段,損傷后預(yù)后較差,肢體功能減退、喪失甚至截肢發(fā)生率高,嚴(yán)重影響患者生活質(zhì)量,因此如何有效促進(jìn)周?chē)窠?jīng)電損傷修復(fù)是目前研究的熱點(diǎn)及難點(diǎn)。膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子(Glial cell line-derived neurotrophic factor,GDNF)是目前發(fā)現(xiàn)的生物活性最強(qiáng)的靶源性神經(jīng)營(yíng)養(yǎng)因子,在損傷后可以減少神經(jīng)元死亡,促進(jìn)神經(jīng)元存活、生長(zhǎng)和分化。脂肪間充質(zhì)干細(xì)胞(adipose-derived stem cells,ADSCs)是目前研究的熱點(diǎn),其多向分化潛能及旁分泌功能對(duì)于促進(jìn)神經(jīng)修復(fù)有著巨大的潛力。本實(shí)驗(yàn)通過(guò)慢病毒轉(zhuǎn)染基因的方法使得ADSCs持續(xù)過(guò)表達(dá)GDNF,觀察其對(duì)于大鼠坐骨神經(jīng)電損傷后肢體運(yùn)功功能恢復(fù)的作用。方法1.采用SD雄性大鼠5只,提取腹股溝脂肪,采用胰酶消化法及差速貼壁法分離并純化大鼠來(lái)源脂肪間充質(zhì)干細(xì)胞,連續(xù)傳代培養(yǎng)至第3代,進(jìn)行分化鑒定及表面標(biāo)記流式細(xì)胞鑒定。2.構(gòu)建過(guò)表達(dá)GDNF的慢病毒,以適當(dāng)?shù)味雀腥続DSCs,觀察感染效率。3.采用SD雄性大鼠33只,建立大鼠坐骨神經(jīng)220V電損傷模型,其中3只用于電損傷病理驗(yàn)證,余30只按隨機(jī)數(shù)字表法分為一個(gè)正常組(6只,常規(guī)飼養(yǎng),不做任何處理),四個(gè)不同處理組(每組6只大鼠):生理鹽水對(duì)照組(NS組),脂肪間充質(zhì)干細(xì)胞組(ADSCs組),膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子組(GDNF組),膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子基因修飾的脂肪間充質(zhì)干細(xì)胞組(GDNF-ADSCs組)。將蛋白溶液或細(xì)胞懸液進(jìn)行損傷神經(jīng)表面注射,等量鹽水對(duì)照處理。觀察時(shí)間8周,每周同一時(shí)間進(jìn)行垂繩積分及后肢平均步幅測(cè)量,記錄結(jié)果。并于第8周最后一天處死,收集傷側(cè)及正常組同側(cè)坐骨神經(jīng),進(jìn)行形態(tài)學(xué)觀察。4.采用SD雄性大鼠120只,每組24只,分組及處理同上。傷后4周處理組取傷側(cè)坐骨神經(jīng),正常組收取同側(cè)神經(jīng)提取組織蛋白,通過(guò)蛋白印跡法對(duì)GDNF蛋白進(jìn)行定量測(cè)定。結(jié)果培養(yǎng)并鑒定了SD大鼠腹股溝脂肪來(lái)源的ADSCs;成功構(gòu)建過(guò)表達(dá)GDNF的慢病毒載體;慢病毒感染ADSCs成功,感染效率高達(dá)95%。垂繩積分平均分顯示GDNF-ADSC是組恢復(fù)趨勢(shì)較好。根據(jù)統(tǒng)計(jì)分析,傷后第8周,GDNF-ADSCs組垂繩積分明顯優(yōu)于NS組,ADSCs組、GDNF組,μ值分別為6.02、2.54、2.67,p0.05或p0.01,余組間比較無(wú)統(tǒng)計(jì)學(xué)差異。NS組、ADSCs組、GDNF組、GDNF-ADSCs組在傷后各時(shí)間點(diǎn)后肢步幅評(píng)分均明顯低于正常組(p值均小于0.05);傷后第3、5、7周,ADSCs組及GDNF組后肢步幅均明顯優(yōu)于NS組(p值均小于0.05),第8周GDNF組后肢步幅優(yōu)于NS組(p值小于0.05);傷后4~8周,GDNF-ADSCs組后肢步幅評(píng)分明顯優(yōu)于其他傷后組(p值均小于0.05)。傷后8周,NS組與其他各組比較有髓纖維數(shù)明顯減少(p值均小于0.05);ADSCs組、GDNF組GDNF-ADSCs組與正常組比較,有髓纖維數(shù)明顯升高,軸突直徑明顯減小,髓鞘厚度明顯增加(p值均小于0.05);GDNF-ADSCs組有髓神經(jīng)纖維數(shù)及髓鞘厚度明顯高于ADSCs組及GDNF組(p值均小于0.05)。傷后4周4個(gè)傷后組GDNF蛋白表達(dá)明顯高于正常組(p值均小于0.05);GDNF-ADSCs組蛋白表達(dá)量明顯高于對(duì)照組、ADSCs組、GDNF組(p值均小于0.05)。結(jié)論1.慢病毒轉(zhuǎn)染脂肪間充質(zhì)干細(xì)胞效率較高,且穩(wěn)定,對(duì)干細(xì)胞正常生長(zhǎng)無(wú)明顯影響。2.過(guò)表達(dá)GDNF的慢病毒轉(zhuǎn)染的ADSCs對(duì)于大鼠坐骨神經(jīng)電損傷后神經(jīng)修復(fù)及肢體運(yùn)動(dòng)功能的恢復(fù)具有更明顯的促進(jìn)作用。3.采用慢病毒轉(zhuǎn)染的手段可以有效改變ADSCs對(duì)GDNF的表達(dá);在傷后4周,經(jīng)GDNF慢病毒載體修飾的ADSCs處理組大鼠損傷神經(jīng)內(nèi)依然穩(wěn)定持續(xù)過(guò)表達(dá)GDNF,其表達(dá)量與最終恢復(fù)效果呈正相關(guān)。
[Abstract]:With the development of modern society, the use of industrial electricity and living electricity is increasingly frequent, and the electrical damage caused by accidental contact in daily production and life is often occurring. As a conductive body, the human body is also called electrical burns. In the same cross section, the higher the resistance part, the higher the heat production, the more obvious the damage to the human body. Therefore, the skin and bone are damaged seriously and the nerve damage is lighter. At the same time, the current entrance of the skin into the body is first contact with the skin in the direction of current conduction, and the part of the outflow and out of the body is the current outlet. The current contact is in contact with the current. At the moment of or out of the skin, the discharge phenomenon may be produced, causing damage to the human body, and the damage degree is even higher than the heat damage. The discharge phenomenon of the current outlet is more obvious, thus the tissue damage is more serious. Other pathological changes, such as vascular embolism, muscle necrosis and neurodegeneration, are still unable to be effectively solved. According to statistics, electrical injury is mainly caused by contact of limbs and many injuries of peripheral nerve. There is no effective repair method for nerve injury in clinic, poor prognosis after injury, loss of limb function, loss or even intercepting. The incidence of limb is high, which seriously affects the quality of life of the patients. Therefore, how to effectively promote the repair of peripheral nerve damage is a hot and difficult point of study. Glial cell line-derived neurotrophic factor (GDNF) is the most bioactive target derived neurotrophic factor, which is found at present, after injury. Adipose-derived stem cells (ADSCs) is a hot topic at present. The multidirectional differentiation potential and paracrine function have great potential for the promotion of nerve repair. In this experiment, the method of gene transfection by lentivirus caused ADSCs to continue. GDNF was used to observe the function of the limb function recovery after the electrical injury of the sciatic nerve in rats. Method 1. 5 male rats of SD were used to extract the fat of the groin. The adipose mesenchymal stem cells were isolated and purified by trypsin digestion and differential adherence, and cultured to third generations for differentiation and surface labeling. Flow cytometry was used to identify the lentivirus expressing GDNF in.2., to infect ADSCs with appropriate titer, and to observe the infection efficiency of.3. in 33 male SD rats, and to establish the 220V electrical injury model of the sciatic nerve of the rat, of which 3 were used for the pathological examination of electrical injury, and the remaining 30 were divided into a normal group by random number table method (6 rats were reared routinely without any treatment). Four different treatment groups (6 rats in each group): normal saline control group (group NS), adipose mesenchymal stem cell group (group ADSCs), glial derived neurotrophic factor group (group GDNF), glial derived neurotrophic factor gene modified adipose mesenchymal stem cell group (group GDNF-ADSCs). Protein solution or cell suspension was used to damage nerve. Surface injection, equal amount of saline control treatment. Observation time 8 weeks, the same time per week to carry out the vertical rope integral and the average hind leg measure, record the results. And on the last day of eighth weeks to death, collect the injured side and the same side of the normal group of the sciatic nerve, observe the morphological observation of.4. SD male rats 120, each group of 24, group and treatment of the same. 4 after injury. 4 The peripheral sciatic nerve was injured in the week treatment group, and the normal group collected the protein of the ipsilateral nerve extract, and the GDNF protein was measured by Western blot. The ADSCs of the fat source of the groin in SD rats was cultured and identified, and the lentivirus vector expressing GDNF was successfully constructed. The slow virus infection ADSCs was successful and the infection efficiency was as high as 95%. vertical rope product. According to statistical analysis, eighth weeks after injury, the vertical rope score of group GDNF-ADSCs was obviously better than that of group NS, ADSCs group, GDNF group, P0.05 or P0.01, respectively, and there was no statistical difference between the other groups.NS group, ADSCs group, GDNF group, and the hind limb stride score at each time point after injury. It was significantly lower than the normal group (P value was less than 0.05), and the hind limb stride of group ADSCs and GDNF group was significantly better than that of group NS (P value was less than 0.05) in group ADSCs and GDNF, and the hind limb stride of GDNF group was better than that of NS group (p less than 0.05) in eighth weeks, and the hind limb stride of the GDNF-ADSCs group was better than that of other groups after injury (P values were less than 0.05). 8 weeks after injury, the group was more than 8 weeks after injury. The number of myelinated fibers in each group was significantly reduced (P value was less than 0.05). In group ADSCs, the number of myelinated fibers increased significantly, the diameter of axon decreased significantly and the thickness of myelin sheath was significantly increased (P value was less than 0.05) compared with the normal group, and the myelinated nerve fiber dimension and the thickness of myelin sheath in group GDNF-ADSCs were significantly higher than that of the ADSCs group and GDNF group (P values were small). The expression of GDNF protein was significantly higher than that of the normal group (P value was less than 0.05) after 4 injuries at 4 weeks after injury. The expression of GDNF-ADSCs protein in group GDNF-ADSCs was significantly higher than that of control group, ADSCs group and GDNF group (P value was less than 0.05). Conclusion 1. lentivirus transfected adipose mesenchymal stem cells were more efficient and stable, and there was no significant effect of.2. over expression GDN on the normal growth of stem cells. The ADSCs transfected with lentivirus of F has a more obvious promoting effect on the nerve repair and the recovery of motor function after the electrical injury of the sciatic nerve in rats..3. can effectively change the expression of ADSCs to GDNF by the means of lentivirus transfection; 4 weeks after the injury, the ADSCs treated group of the GDNF lentivirus carrier is still stable in the injured nerve. The expression level of GDNF was positively correlated with the final recovery.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R647

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