Effect of rat platelet-rich plasma gel on autologous adipose-derived mesenchymal stem cells overexpressing glia-derived neurotrophic factor
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摘要:
目的 探讨大鼠富血小板血浆（PRP）凝胶对过表达胶质细胞源性神经营养因子（GDNF）的自体脂肪间充质干细胞（ADSC）的作用。 方法 该研究为实验研究。取5只成年雄性SD大鼠,采用胶原酶消化法获取原代ADSC并成功鉴定。取第3代ADSC,按照随机数字表法（分组方法下同）分为感染空载腺病毒的阴性对照组和感染过表达GDNF腺病毒的过表达GDNF组。培养48 h后,大体观察细胞感染情况。另取5只成年雄性SD大鼠,采血后采用差速离心法获取PRP,制备成凝胶后采用扫描电子显微镜观察其微观结构。取第3代ADSC,加入PRP成胶前的混合液中,成胶后常规培养48 h,行苏木精-伊红染色和钙黄素/碘化丙啶染色观测细胞生长情况。将感染空载腺病毒的ADSC或感染过表达GDNF腺病毒的ADSC在PRP凝胶中进行常规培养。培养48 h后,采用钙黄素/碘化丙啶染色检测细胞生长情况；培养24、48、72 h及1、2、3、4周后,收集细胞培养基的上清液,采用酶标仪测定其吸光度值并计算GDNF含量,样本数为3；培养48 h后,采用免疫荧光法检测细胞表达施万细胞特异性标志物S100蛋白质的情况。 结果 培养48 h后,阴性对照组和过表达GDNF组中感染腺病毒的细胞占比均接近90%且生长状态良好。阴性对照组细胞正常生长；过表达GDNF组细胞的形态发生明显变化,80%~90%的细胞伸出2个或多个突起,且在细胞聚集的地方,突起交织成网。PRP凝胶呈三维网状结构,且孔径大小不一。培养48 h后,ADSC可以很好地附着于PRP凝胶,且活细胞占比达98%。培养48 h后,感染空载腺病毒的ADSC生长状态良好,且呈典型的ADSC样梭形生长；感染过表达GDNF腺病毒的ADSC生长状态良好,且多数细胞伸出2个或多个突起,突起聚集处交织成网。培养24、48、72 h及1、2、3、4周后,感染过表达GDNF腺病毒的ADSC培养基的上清液中GDNF含量分别为（90±10）、（133±15）、（150±10）、（137±15）、（132±18）、（120±10）、（127±16）pg/mL,均明显高于感染空载腺病毒的ADSC的（42±7）、（44±7）、（43±6）、（47±6）、（49±5）、（49±6）、（51±4）pg/mL,t值分别为6.20、8.08、15.18、9.12、7.99、9.61、7.86,P<0.05。培养48 h后,与感染空载腺病毒的ADSC相比,感染过表达GDNF腺病毒的ADSC中S100蛋白质表达明显增强。 结论 制备的自体三维PRP凝胶具有良好的生物相容性,可负载过表达GDNF的大鼠ADSC并缓释GDNF,诱导ADSC向高表达S100蛋白质的施万细胞分化。 Abstract:Objective To investigate the effect of rat platelet-rich plasma (PRP) gel on autologous adipose-derived mesenchymal stem cells (ADSCs) overexpressing glial-derived neurotrophic factor (GDNF). Methods This study was an experimental study. Five adult male Sprague-Dawley rats were used, and the primary ADSCs were obtained by collagenase digestion, and then the cells were identified successfully. The 3rd passage of ADSCs were obtained and divided into negative control group infected with unloaded adenovirus and overexpressing GDNF group infected with overexpressing GDNF adenovirus, according to random number table method (the grouping method was the same below). After 48 hours of culture, the infection of cells was observed. Five adult male Sprague-Dawley rats were used, and the PRP was obtained after collecting blood by differential centrifugation. PRP was prepared into a gel and its microstructure was observed by scanning electron microscope. The ADSCs of 3rd passage were added into the PRP solution mixture and cultured for 48 hours after gelation. The cell growth was observed by hematoxylin-eosin staining and calcein/propyl iodide staining. ADSCs infected with unloaded adenovirus and ADSCs infected with overexpressing GDNF adenovirus were routinely cultured in PRP gel. After 48 hours of culture, the cell growth was detected by calcein/propyl iodide staining. After culture for 24, 48, 72 hours and 1, 2, 3, 4 weeks, the supernatant of cell culture medium was collected, the absorbance value was determined by microplate analyzer, and the GDNF content was calculated, with the sample number of 3. After 48 hours of culture, the expression of S100 protein (a specific marker of Schwann cells) was detected by immunofluorescence assay. Results After 48 hours of culture, the proportions of cells infected with adenovirus in negative control group and overexpressing GDNF group were close to 90%, and the cell growth was good. The cells in negative control group grew normally. The morphology of the cells in overexpressing GDNF group changed significantly with 80%-90% of the cells having two or more protrusions, and the protrusions were interwoven to form a network wherever the cells gathered. PRP gel formed a three-dimensional network structure with different pore sizes. After 48 hours of culture, ADSCs could be well attached to PRP gel, and 98% of the cells were alive. After 48 hours of culture, ADSCs infected with unloaded adenovirus grew well and showed typical ADSC-like spindle-shaped growth. ADSCs infected with overexpressing GDNF adenovirus grew well, and most of the cells had two or more protrusions, and the protrusions were interwoven into a network. After culture for 24, 48, 72 hours and 1, 2, 3, 4 weeks, the content of GDNF in the supernatant of ADSCs infected with overexpressing GDNF adenovirus was (90±10), (133±15), (150±10), (137±15), (132±18), (120±10), and (127±16) pg/mL, which was significantly higher than (42±7), (44±7), (43±6), (47±6), (49±5), (49±6), and (51±4) pg/mL of ADSCs infected with unloaded adenovirus (with t values of 6.20, 8.08, 15.18, 9.12, 7.99, 9.61, and 7.86, respectively, P<0.05). After 48 hours of culture, the fluorescence intensity of S100 protein expression of ADSCs infected with overexpressing GDNF adenovirus was significantly stronger than that of ADSCs infected with unloaded adenovirus. Conclusions The prepared autologous three-dimensional PRP gel has good biocompatibility and can carry rat GDNF-overexpressing ADSCs and release GDNF slowly, inducing ADSCs to differentiate into Schwann cells that express high level of S100 protein. -
参考文献
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图 1 原代大鼠脂肪间充质干细胞（ADSC）的鉴定 倒置相差显微镜×40。1A.原代ADSC培养48 h后呈典型的漩涡样排列；1B.第3代ADSC成脂诱导分化14 d后,细胞质内有大量遮光性强的红色脂滴；1C.第3代ADSC成骨诱导分化21 d后,细胞膜表面、细胞质中均可见大量红色或紫红色颗粒状钙化结节
图 2 第3代大鼠脂肪间充质干细胞感染腺病毒48 h后的生长情况 荧光显微镜×40。2A、2B、2C.分别为阴性对照组细胞感染及生长情况的荧光、非荧光、复合图,绝大多数细胞感染了腺病毒且生长状态良好；2D、2E、2F.分别为过表达GDNF组细胞感染及生长情况的荧光、非荧光、复合图,绝大多数细胞感染了腺病毒,且多数细胞有2个或多个突起
注：GDNF为胶质细胞源性神经营养因子；阴性对照组、过表达GDNF组细胞分别感染空载腺病毒和过表达GDNF的腺病毒
图 3 富血小板血浆凝胶的微观结构及其中的单纯大鼠脂肪间充质干细胞在培养48 h后的生长情况。3A.凝胶呈三维网状,孔径大小不一 扫描电子显微镜×50；3B.细胞（蓝色箭头指示）可以附着在凝胶中生长 苏木精-伊红×20；3C.生长在凝胶中的绝大多数细胞为活细胞（绿色）,死细胞（红色）很少 钙黄绿素-碘化丙啶×40
图 4 2组感染腺病毒的大鼠脂肪间充质干细胞（ADSC）在富血小板血浆凝胶中培养48 h后的生长情况 绿色荧光蛋白×40。4A.感染空载腺病毒的ADSC细胞呈典型的ADSC样梭形生长；4B.感染过表达GDNF腺病毒的ADSC的多数细胞伸出2个或多个突起,突起聚集处交织成网状
注：成功感染腺病毒的细胞呈绿色
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