本文選題：神經(jīng)修復(fù) + 神經(jīng)營(yíng)養(yǎng)因子��； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景和意義 神經(jīng)母細(xì)胞瘤細(xì)胞(SHSY5Y)是神經(jīng)系統(tǒng)疾病研究中重要的細(xì)胞系,該細(xì)胞表達(dá)神經(jīng)營(yíng)養(yǎng)因子。本實(shí)驗(yàn)制備的SHSY5Y提取液是此細(xì)胞內(nèi)的蛋白,對(duì)中樞和外周神經(jīng)系統(tǒng)有營(yíng)養(yǎng)作用。干細(xì)胞療法是研究組織修復(fù)的重要方法。臍帶間充質(zhì)干細(xì)胞(UC-MSCs)是一類(lèi)具有多向分化潛能和自我更新能力的細(xì)胞群。UC-MSCs可分泌細(xì)胞因子,如神經(jīng)生長(zhǎng)因子(NGF)、腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)等,在神經(jīng)損傷后的修復(fù)過(guò)程中發(fā)揮重要作用。研究表明增加損傷部位神經(jīng)營(yíng)養(yǎng)因子可促進(jìn)神經(jīng)修復(fù)。 本實(shí)驗(yàn)用SHSY5Y提取液處理UC-MSCs,檢測(cè)處理后細(xì)胞分泌NGF和BDNF的變化,觀察處理后的干細(xì)胞對(duì)坐骨神經(jīng)損傷的促修復(fù)作用,為周?chē)窠?jīng)損傷修復(fù)提供一種有益的思路。 材料與方法 應(yīng)用超聲破碎法得到SHSY5Y提取液,BCA法進(jìn)行蛋白定量。復(fù)蘇UC-MSCs,采用無(wú)血清培養(yǎng)基接種至6孔板,105/孔。在實(shí)驗(yàn)組的UC-MSCs中加入SHSY5Y細(xì)胞提取液,濃度為50ul/ml誘導(dǎo)7天。7天后分別檢測(cè)兩組細(xì)胞的表面標(biāo)志物；分別檢測(cè)目的基因BDNF和NGF在兩組細(xì)胞中的表達(dá)情況。采用大鼠坐骨神經(jīng)擠壓損傷動(dòng)物模型,36只SD雌性大鼠,隨機(jī)分成6組(A-F),分別是生理鹽水組,SHSY5Y提取液組,MSC組,SHSY5Y提取液和MSC組,誘導(dǎo)后的MSC組和正常對(duì)照組。術(shù)后觀察各組大鼠肢體狀況：步態(tài)變化,足趾攣縮、肌肉萎縮及恢復(fù)情況。參照沈江寧的足跡分析法,采用Brain公式計(jì)算坐骨神經(jīng)功能指數(shù)(SFI)。術(shù)后3周檢測(cè)大鼠小腿三頭肌萎縮恢復(fù)情況,測(cè)定肌濕重恢復(fù)率(術(shù)側(cè)小腿三頭肌濕重/正常對(duì)照側(cè)×100%),以評(píng)價(jià)再生神經(jīng)對(duì)靶組織的支配程度。分析靶肌肉和神經(jīng)病理學(xué)HE染色組織切片。 結(jié)果 1.應(yīng)用膠原酶消化法分離臍帶間充質(zhì)干細(xì)胞,應(yīng)用超聲破碎法得到SHSY5Y提取液； 2.培養(yǎng)對(duì)照組和實(shí)驗(yàn)組兩組細(xì)胞,對(duì)兩組細(xì)胞分別進(jìn)行細(xì)胞表型鑒定,實(shí)驗(yàn)組MSC的CD29, CD44, CD105和HLA-ABC結(jié)果顯示仍為陽(yáng)性； 3.分別提取兩組細(xì)胞總RNA進(jìn)行RT-PCR,實(shí)驗(yàn)組NGF和BDNF均有一定增加； 4.制作坐骨神經(jīng)擠壓損傷模型,結(jié)果顯示大鼠肌肉痙攣緩解程度均優(yōu)于生理鹽水對(duì)照組； 5.坐骨神功能指數(shù)測(cè)定結(jié)果顯示各實(shí)驗(yàn)治療組與生理鹽水組之間均有顯著差異(P0.05)； 6.小腿三頭肌濕重恢復(fù)率測(cè)定結(jié)果顯示各治療組與生理鹽水組間對(duì)比有顯著差異(P0.05); 7.術(shù)后3w取術(shù)側(cè)小腿三頭肌及損傷處坐骨神經(jīng)做橫切片HE染色,顯示誘導(dǎo)后MSC組肌肉只有極少量的中性粒細(xì)胞,相對(duì)其他各組其神經(jīng)髓鞘恢復(fù)較好。 結(jié)論1. SHSY5Y提取液可使UC-MSCs分泌的NGF和BDNF核酸表達(dá)量有所增加；2. SHSY5Y提取液誘導(dǎo)的UC-MSCs能夠更好地促進(jìn)坐骨神經(jīng)損傷后的修復(fù)。
[Abstract]:Background and significance Neuroblastoma cell line SHSY 5Y is an important cell line in the study of nervous system diseases, which expresses neurotrophic factor. The SHSY5Y extract prepared in this experiment is the protein in this cell and has nutritional effects on the central and peripheral nervous system. Stem cell therapy is an important method to study tissue repair. Umbilical cord mesenchymal stem cells (UC-MSCs) are a group of cells with multidirectional differentiation potential and self-renewal ability. UC-MSCs secrete cytokines, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and so on. It plays an important role in the repair of nerve injury. It has been shown that increasing neurotrophic factor in injured site can promote nerve repair. In this experiment, UC-MSCs were treated with SHSY5Y extract, the changes of NGF and BDNF secreted by the treated cells were detected, and the effects of the treated stem cells on the repair of sciatic nerve injury were observed, which provided a useful way of thinking for the repair of peripheral nerve injury. Materials and methods The SHSY5Y extract was obtained by ultrasonic crushing method for protein quantification. UC-MSCs were resuscitated and inoculated with serum-free medium to 6 hole plate (10 5 / well). After 7 days of 50ul/ml induction, the surface markers of the two groups of cells were detected, and the expression of the target gene BDNF and NGF in the two groups were detected respectively. Thirty-six SD female rats were randomly divided into 6 groups: normal saline group, SHSY5Y extract group, MSC group and normal control group. The changes of gait, toe contracture, muscle atrophy and recovery were observed after operation. According to Shen Jiangning's footprint analysis, the sciatic nerve function index (SFI) was calculated by Brain formula. Three weeks after operation, the recovery of triceps muscle atrophy and the recovery rate of muscle wet weight (wet weight of triceps muscle / normal control side 脳 100) were measured to evaluate the degree of regenerative nerve innervation to target tissue. The HE staining sections of target muscle and neuropathology were analyzed. Result 1. Umbilical cord mesenchymal stem cells were isolated by collagenase digestion and SHSY5Y extract was obtained by ultrasonic fragmentation. 2. The cell phenotypes of the control group and the experimental group were identified respectively. The results of CD29, CD44, CD105 and HLA-ABC of MSC in the experimental group were still positive. 3. The total RNA of the two groups were extracted for RT-PCR.The NGF and BDNF of the experimental group were increased to a certain extent. 4. The model of sciatic nerve crush injury was made. The results showed that the remission degree of muscle spasm in rats was better than that in normal saline control group. 5. The results of ischium function index test showed that there was significant difference between the experimental treatment group and normal saline group (P 0.05). 6. The results showed that there was significant difference between the treatment group and the normal saline group (P 0.05). 7. Three weeks after operation, the triceps muscle of the leg and the sciatic nerve were stained with HE. The results showed that there were only a few neutrophils in the muscles of the MSC group after induction, which was better than that of the other groups. Conclusion 1. SHSY5Y extract could increase the expression of NGF and BDNF nucleic acids secreted by UC-MSCs. UC-MSCs induced by SHSY5Y extract can promote the repair of sciatic nerve injury.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.4

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