發(fā)布時(shí)間：2018-09-12 10:18

【摘要】：目的本研究旨在應(yīng)用脫細(xì)胞的方法將細(xì)胞完全去除,成功制作出大鼠脫細(xì)胞脊髓支架,并且最大限度的保留了脊髓細(xì)胞外基質(zhì)的空間三維立體結(jié)構(gòu)。將制備好的脫細(xì)胞脊髓支架磨碎后用生理鹽水溶解制成脫細(xì)胞脊髓支架顆粒懸濁液,通過大鼠尾靜脈注射,來評(píng)估其在大鼠體內(nèi)的安全性,從而為脫細(xì)胞脊髓支架應(yīng)用于治療脊髓損傷提供依據(jù)。方法1.取Wistar大鼠4只,麻醉后手術(shù)取出脊髓,采用脫細(xì)胞方法將脊髓中神經(jīng)細(xì)胞脫掉后,制作出脫細(xì)胞支架。將制備的支架進(jìn)行HE染色及髓鞘染色光鏡下觀察脫細(xì)胞脊髓組織結(jié)構(gòu),驗(yàn)證大鼠脊髓支架是否成功脫掉細(xì)胞成分。2.取制備的脫細(xì)胞脊髓支架,磨碎后放入500ml生理鹽水中,制成脫細(xì)胞脊髓支架懸濁液。取Wistar大鼠48只,平均分成2組,一組為實(shí)驗(yàn)組,另一組為對(duì)照組,每組24只,再分為6個(gè)時(shí)間節(jié)點(diǎn),每個(gè)時(shí)間節(jié)點(diǎn)4只,分別對(duì)應(yīng)1天、2天、3天、7天、10天、14天,實(shí)驗(yàn)組每只大鼠經(jīng)尾靜脈注射脫細(xì)胞脊髓懸濁液2ml,對(duì)照組每只大鼠經(jīng)尾靜脈注射生理鹽水2ml,分別于尾靜脈注射后1天、2天、3天、7天、10天、14天取血后處死,檢測(cè)血清中谷丙轉(zhuǎn)氨酶(ALT)和肌酐(CR)含量,進(jìn)行統(tǒng)計(jì)學(xué)分析,處死的大鼠分別取肺臟、肝臟、脾臟、心臟及腎臟制作成組織切片進(jìn)行HE染色后觀察其組織學(xué)變化。結(jié)果1.采用化學(xué)萃取法制備的Wistar大鼠脫細(xì)胞脊髓支架,呈白色,短柱狀,大小有所減小,觸之質(zhì)地柔韌。HE染色、髓鞘染色顯示成功制備的支架由直徑不同的孔隙組成,細(xì)胞已徹底脫掉,已不含細(xì)胞成分,髓鞘已被脫除,各個(gè)孔洞的壁由細(xì)胞外基質(zhì)組成。2.實(shí)驗(yàn)組大鼠經(jīng)尾靜脈注射脫細(xì)胞脊髓懸濁液后,肺部出現(xiàn)明顯的炎癥反應(yīng)和增生表現(xiàn)。實(shí)驗(yàn)組肝細(xì)胞較對(duì)照組腫脹,相鄰細(xì)胞之間邊界顯示不清,胞核變大,胞漿相對(duì)松散,表現(xiàn)為空泡變性。此現(xiàn)象在經(jīng)尾靜脈注射后第1天較重,隨時(shí)間延長不斷減輕,到第10天肝細(xì)胞恢復(fù)到正常形態(tài)。通過測(cè)定血液中谷丙轉(zhuǎn)氨酶(ALT)的含量,發(fā)現(xiàn)前兩天谷丙轉(zhuǎn)氨酶含量處在較高水平,之后逐漸降低到正常值。腎臟病理檢查未見明顯改變,血清學(xué)檢查發(fā)現(xiàn)肌酐(CR)值在實(shí)驗(yàn)組與對(duì)照組之間無差異。實(shí)驗(yàn)組大鼠脾臟組織切片表現(xiàn)為淋巴小結(jié)較對(duì)照組增大,邊緣區(qū)界限模糊,這種現(xiàn)象隨著時(shí)間的延長而逐漸減輕。實(shí)驗(yàn)組心臟組織切片無顯著異常。結(jié)論1.本實(shí)驗(yàn)采用化學(xué)萃取的方法脫掉脊髓中的細(xì)胞成分,成功制作出大鼠脫細(xì)胞脊髓支架,將細(xì)胞成分徹底脫除,并且最大限度的保留了脊髓細(xì)胞外基質(zhì)的空間三維立體結(jié)構(gòu)。2.將脫細(xì)胞脊髓支架懸濁液經(jīng)大鼠尾靜脈注射后,對(duì)大鼠體內(nèi)部分器官存在一過性的毒性反應(yīng),對(duì)肝臟組織形態(tài)和肝功能以及肺的組織形態(tài)具有一過性損害,在脾臟一過性引發(fā)由B細(xì)胞參與的體液免疫,對(duì)心臟細(xì)胞的組織形態(tài)無損害,對(duì)腎臟的組織形態(tài)及腎功能無損傷。相對(duì)傳統(tǒng)的生物材料支架,脫細(xì)胞脊髓支架具有較好的生物相容性、機(jī)械強(qiáng)度及形態(tài)。為將來進(jìn)行脫細(xì)胞脊髓支架移植治療脊髓損傷提供了依據(jù)。
[Abstract]:Objective To prepare acellular spinal cord scaffolds by acellular method and to preserve the three-dimensional structure of extracellular matrix of spinal cord. To evaluate the safety of acellular spinal stent in rats by tail vein injection, so as to provide basis for the application of acellular spinal stent in the treatment of spinal cord injury. Methods 1. Four Wistar rats were taken out of spinal cord after anesthesia. The acellular scaffold was made after the nerve cells in spinal cord were removed by acellular method. E staining and myelin sheath staining were used to observe the structure of acellular spinal cord and to verify whether the acellular spinal cord scaffolds were successfully removed. 2. The acellular spinal cord scaffolds were prepared and ground into 500 ml normal saline to form acellular spinal cord scaffold suspension. The control group, 24 rats in each group, were divided into 6 time nodes, each time node 4, corresponding to 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, the experimental group each rat through the tail vein injection of acellular spinal cord suspension 2 ml, the control group each rat through the tail vein injection of normal saline 2 ml, respectively in the tail vein injection 1 day, 2 days, 3 days, 7 days, 10 days, 14 days to take Serum ALT and CR levels were measured and analyzed statistically. Lung, liver, spleen, heart and kidney were taken from the sacrificed rats and stained with HE. The histological changes were observed. Results 1. The acellular spinal cord scaffold of Wistar rats prepared by chemical extraction was white and short. HE staining and myelin sheath staining showed that the scaffolds were composed of pores with different diameters, the cells had been completely removed, and the myelin sheath had been removed. The walls of the holes were composed of extracellular matrix. 2. After injection of acellular spinal cord suspension into the tail vein, the rats in the experimental group were pulmonary. The hepatocytes in the experimental group were swollen, the boundary between adjacent cells was unclear, the nucleus was enlarged and the cytoplasm was relatively loose, showing vacuolar degeneration. Glutamic-pyruvic aminotransferase (ALT) levels in the blood were determined, and the ALT levels were higher two days ago, and then gradually decreased to normal values. There was no significant abnormality in the cardiac tissue sections of the experimental group. Conclusion 1. The acellular spinal cord scaffold was successfully made by chemical extraction, and the acellular spinal cord scaffold was completely removed and preserved to the maximum extent. The three-dimensional structure of spinal extracellular matrix was preserved. 2. After the acellular spinal cord scaffold suspension was injected into the tail vein of rats, transient toxic reactions were found in some organs of rats, which caused transient damage to liver morphology, liver function and lung morphology, and B cells participated in the transient damage of spleen. Compared with traditional biomaterial scaffolds, acellular spinal cord scaffolds have better biocompatibility, mechanical strength and morphology, which provide a basis for the future treatment of spinal cord injury with acellular spinal cord scaffolds.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2

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