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Title: [Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis].
Author: Chen J, Wang N, Zhang H, Zhang X, Zhao L, Zhu L, Li Z, Bei C.
Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2020 Nov 15; 34(11):1438-1445. PubMed ID: 33191703.
Abstract:
OBJECTIVE: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. METHODS: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. RESULTS: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). CONCLUSION: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration. 目的: 探索沉默 P75 神经营养蛋白受体（p75 neurotrophin receptor，P75NTR）联合 NGF 过表达对大鼠 BMSCs 增殖活性和复合脱钙骨基质（demineralized bone matrix，DBM）构建组织工程骨异位成骨能力的影响。. 方法: 通过贴壁分离法培养 SD 大鼠 BMSCs 并传代。取第 3 代 BMSCs，分别用慢病毒介导沉默 P75NTR 基因（B 组）、NGF 过表达基因（C 组）、沉默 P75NTR 和 NGF 过表达双基因（D 组）转染，以未转染细胞作为对照（A 组）。转染 7 d 后荧光显微镜观察目的基因荧光蛋白表达；细胞计数试剂盒 8 法检测转染后连续 8 d 细胞的活性；Western blot 检测各组 P75NTR 和 NGF 蛋白表达。倒置相差显微镜和扫描电镜分别观察沉默 P75NTR 和 NGF 过表达双基因转染后 BMSCs 与 DBM 的黏附情况。将上述 4 组转染后 BMSCs 分别与 DBM 共培养制备组织工程骨后，埋植于 8 周龄 SD 大鼠背侧皮下构建皮下异位成骨模型（ n=6），术后 4、8 周行 HE 染色，术后 8 周 ALP 染色观察钙结节形成，实时荧光定量 PCR 检测成骨相关基因 Runt 相关转录因子 2（Runt-related transcription factor 2，Runx2）、ALP 和骨钙素（osteocalcin，OCN）表达。. 结果: 转染 7 d A 组未见荧光表达，B 组呈红色荧光表达，C 组呈绿色荧光表达，D 组呈红绿色复合荧光表达；目的基因荧光表达率可达 70% 左右。Western blot 检测示，A、C 组 P75NTR 蛋白相对表达量显著高于 B、D 组，C、D 组 NGF 蛋白相对表达量显著高于 A、B 组，差异均有统计学意义（ P<0.05）。随时间推移，各组细胞增殖活性均上升，其中 D 组上升最明显，3～8 d 细胞活性明显高于 A 组（ P<0.05）。倒置相差显微镜和扫描电镜观察示，BMSCs 能与 DBM 形成良好黏附。皮下异位成骨实验中，HE 染色示术后 4、8 周，D 组比其余 3 组有更多的骨组织形成。ALP 染色示 D 组 ALP 活性最高，有更好的成骨表达。实时荧光定量 PCR 检测示，与 A 组比较，D 组 Runx2、ALP、OCN mRNA 相对表达量显著升高，差异均有统计学意义（ P<0.05）。. 结论: 沉默 P75NTR 联合 NGF 过表达双基因共转染 BMSCs 复合 DBM 构建组织工程骨具有良好的异位成骨能力，通过提高 NGF 的水平、关闭 P75NTR 凋亡通道，不仅可以提高细胞活性，还能更好地促进骨组织再生。.

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