敲除LRRK2基因的PC12细胞移植对帕金森病大鼠的疗效  

Effect of PC12 Cell Transplantation Based on CRISPR/Cas9 Knockout of LRRK2 Gene in Parkinson′s Disease Rats

作　　者：刘治华 韦丽萍 田慧虹 蔡慧敏 聂坤[2] 张玉虎[2] 王丽娟[2] 王丽敏 LIU Zhihua;WEI Liping;TIAN Huihong(School of Medicine,South China University of Technology,Guangdong 510006,China)

机构地区：[1]华南理工大学医学院,广州510006 [2]广东省人民医院,广州510080

出　　处：《医学研究杂志》2023年第5期83-88,共6页Journal of Medical Research

基　　金：广东省自然科学基金资助项目(2018A030313427);广东省广州市科技计划项目(202002030393)。

摘　　要：目的探究经CRISPR/Cas9单载体慢病毒敲除LRRK2基因的PC12细胞移植至帕金森病模型大鼠右侧纹状体的治疗效果及作用机制。方法用CRISPR/Cas9单载体慢病毒转染PC12细胞构建敲除LRRK2基因的细胞株,Western blot法检测敲除效率;颈背部皮下注射鱼藤酮构建帕金森病大鼠模型,通过立体定向注射移植敲除LRRK2基因的PC12细胞至帕金森病大鼠右侧纹状体,移植1周后采用旷场实验、爬杆实验检测对大鼠的运动功能的改变,用Western blot法、免疫组化检测纹状体区凋亡相关蛋白caspase-3、Bcl-2、Bax以及酪氨酸羟化酶(tyrosine hydroxylase,TH)含量的变化探索经CRISPR/Cas9单载体慢病毒敲除LRRK2基因的PC12细胞对PD模型大鼠运动症状改善的作用机制。结果右侧纹状体PC12细胞移植治疗1周后,与帕金森病组大鼠比较,移植敲除LRRK2基因的PC12细胞的PD大鼠的旷场实验运动总路程延长、运动平均速度增大及爬杆试验评分降低(P<0.05);右侧纹状体中TH的表达量增多(P<0.05);右侧状体区中细胞凋亡的发生率降低(P<0.05),且上述指标的改善程度均高于移植正常PC12细胞的帕金森病大鼠(P<0.05)。结论经过LRRK2敲除的PC12细胞移植后对帕金森病模型大鼠的运动功能有更好的疗效,其潜在的作用机制可能是减少移植区中细胞凋亡的发生率来增强其疗效,基因修饰LRRK2的细胞移植有望成为帕金森病运动功能的治疗手段之一。Objective To investigate the therapeutic effect and mechanism of transplantation of PC12 cells with LRRK2 gene knocked out by CRISPR/Cas9 lentivirus into the right striatum of Parkinson′s disease(PD)rats.Methods We used CRISPR/Cas9 lentivirus to construct a PC12 cell line with LRRK2 gene knockout,and Western blot to detect the knockout efficiency;to establish the rat model of PD,we used rotenone by subcutaneous injection into the back.Then LRRK2 knockout PC12 cells were transplanted into the right striatum of PD rats by stereotactic injection.One week after transplantation,we used open field test and pole test to detect the changes of motor function,and the apoptosis related proteins caspase-3,Bcl-2 and Bax in striatum were detected by Western blot and immunohistochemistry,aiming to explore the mechanism of LRRK2 knockout PC12 cells on the improvement of motor symptoms in PD rats.Results One week after transplantation,compared with the rats in PD group,the PD rats transplanted with PC12 cells knockout of the LRRK2 gene had longer total distance and higher average speed in open field test,and lower score in pole test(P<0.05);the expression in the right striatum increased(P<0.05);the incidence of apoptosis in the right striatum area decreased(P<0.05),and the improvement of the above indexes was higher than that of PD rats transplanted with normal PC12 cells(P<0.05).Conclusion The transplantation of LRRK2 knockout PC12 cells had a better effect on improving the motor function of PD model rats.The reduced incidence of apoptosis in the transplantation area may be its potential mechanism.The cell transplantation with genetically modified LRRK2 may become one of the treatments for motor function in PD.

关 键 词：LRRK2 基因敲除 帕金森病 细胞凋亡 

分 类 号：R741[医药卫生—神经病学与精神病学]