负载microRNA-27b-骨髓间充质干细胞来源外泌体的软骨细胞-聚乳酸羟基乙酸共聚物骨软骨复合体移植治疗软骨缺损的实验研究

目的:探讨负载microRNA-27b(miR-27b)-骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)来源外泌体(exosomes,exo)的软骨细胞(chondrocytes,chon)-聚乳酸羟基乙酸共聚物poly(lactic-co-glycolic acid),PLGA]骨软骨复合体移植治疗软骨缺损的效果及作用机制。方法:(1)培养BMSC并制备BMSC来源exo(BMSC-exo),观察BMSC-exo的形态,测定BMSC-exo的粒径和Zeta电位,并采用蛋白质印迹法(Western Blot)检测BMSC-exo表面标记蛋白CD9和CD63。(2)将培养至第2或第3代的BMSC分为2组,BMSC组于RPMI-1640培养基中培养,不进行干预,miR-27b-BMSC组采用miR-27b过表达的重组腺病毒感染;培养24 h后,制备BMSC-exo和miRNA-27b-BMSC-exo,并分别提取BMSC、BMSC-exo、miRNA-27b-BMSC、miRNA-27b-BMSC-exo的RNA,采用实时定量PCR检测miR-27b的表达。(3)分别采用Dil细胞膜红色荧光探针和Hoechst33258染色试剂给miR-27b-BMSC-exo和大鼠软骨细胞染色,于荧光显微镜下观察大鼠软骨细胞对miR-27b-BMSC-exo的摄取情况。(4)将大鼠软骨细胞分为2组,BMSC-exo组接种于BMSC-exo终浓度为80μg·mL^-1的RPMI-1640培养基中,miR-27b-BMSC-exo组接种于miR-27b-BMSC-exo终浓度为80μg·mL^-1的RPMI-1640培养基中;培养4 h后,采用实时定量PCR检测miR-27b的表达。(5)将大鼠软骨细胞分为4组,对照组于RPMI-1640培养基中培养,不进行干预,白细胞介素(interleukin,IL)-1β组在IL-1β终浓度为10 ng·mL^-1的RPMI-1640培养基中培养,miR-27b-BMSC-exo组在IL-1β终浓度为10 ng·mL^-1、miR-27b-BMSC-exo终浓度为80μg·mL^-1的RPMI-1640培养基中培养,BMSC-exo组在IL-1β终浓度为10 ng·mL^-1、BMSC-exo终浓度为80μg·mL^-1的RPMI-1640培养基中培养;分别于培养0 h、1 h、2 h、3 h、4 h和8 h,采用MTT法测定各组大鼠软骨细胞活力;培养24 h后,提取各组大鼠软骨细胞总蛋白,采用Western Blot检测软骨损伤相关蛋白半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid specific protease,caspase)-3、caspase-9、基质金属蛋白酶(matrix metalloproteinase,MMP)-13的表达。(6)取18只8周龄SD雌性大鼠,于SD大鼠的左后肢股骨远端滑车沟槽制造直径4.5 mm、深度1 mm的软骨缺损,建立软骨缺损SD大鼠模型;将18只软骨缺损SD大鼠模型随机分为3组,对照组植入chon-PLGA骨软骨复合体,miR-27b-BMSC-exo组植入miR-27b-BMSC-exo-chon-PLGA骨软骨复合体,BMSC-exo组植入BMSC-exo-chon-PLGA骨软骨复合体;饲养12周后处死SD大鼠,观察软骨修复效果,免疫组织化学染色检测软骨损伤修复标志蛋白Ⅱ型胶原蛋白、MMP-13的表达;提取SD大鼠软骨组织总蛋白,采用Western Blot检测软骨损伤相关蛋白caspase-3、caspase-9、MMP-13的表达。结果:(1)BMSC-exo的鉴定结果。BMSC-exo为圆盘形囊泡状膜结构,粒径92~115 nm,数量占比最高的BMSC-exo的粒径为106 nm;BMSC-exo的Zeta电位为(-22.13±2.1)mV。BMSC-exo表面标记物CD9和CD63在BMSC-exo组的表达量显著高于BMSC组(相对灰度值:6.513±0.714,1.001±0.021,t=18.902,P=0.000;7.564±0.636,1.026±0.027,t=25.158,P=0.000)。(2)miR-27b表达的检测结果。miR-27b在miR-27b-BMSC组BMSC中的表达量高于BMSC组(相对表达量:46.785±8.153,1.000±0.280,t=13.748,P=0.000);miR-27b在miR-27b-BMSC组exo中的表达量高于BMSC组(相对表达量:34.825±8.612,1.000±0.325,t=9.621,P=0.000)。(3)miR-27b-BMSC-exo的摄取情况。大鼠软骨细胞和miR-27b-BMSC-exo共培养4 h,miR-27b-BMSC-exo被大鼠软骨细胞摄取。(4)共培养后大鼠软骨细胞中miR-27b表达的检测结果。miR-27b在miR-27b-BMSC-exo组大鼠软骨细胞中的表达量高于BMSC-exo组(相对表达量:3.315±0.523,1.000±0.244,t=9.826,P=0.000)。(5)大鼠软骨细胞活力测定结果。时间因素与分组因素存在交互效应(F=2.836,P=0.049)。4组大鼠软骨细胞活力总体比较,差异有统计学意义,即存在分组效应(F=11.345,P=0.049)。培养前及培养1 h、2 h,大鼠软骨细胞活力的组间差异无统计学意义(F=0.047,P=0.406;F=0.765,P=0.189;F=2.095,P=0.063);培养3 h、4 h、8 h,大鼠软骨细胞活力的组间差异有统计学意义(F=4.720,P=0.039;F=7.421,P=0.021;F=95.348,P=0.000),IL-1β组的大鼠软骨细胞活力小于对照组、miR-27b-BMSC-exo组、BMSC-exo组(3 h:P=0.002,P=0.011,P=0.026;4 h:P=0.002,P=0.002,P=0.006;8 h:P=0.004,P=0.004,P=0.011),miR-27b-BMSC-exo组的大鼠软骨细胞活力大于对照组和BMSC-exo组(3 h:P=0.019,P=0.023;4 h:P=0.015,P=0.002;8 h:P=0.003,P=0.029)。不同时间点大鼠软骨细胞活力总体比较,差异无统计学意义,即不存在时间效应(F=0.258,P=0.083)。(6)大鼠软骨细胞中软骨损伤相关蛋白表达的检测结果。切割-caspase-3、切割-caspase-9和MMP-13的表达量组间比较,差异有统计学意义(F=15.691,P=0.024;F=98.021,P=0.002;F=76.312,P=0.004),IL-1β组大鼠软骨细胞中切割-caspase-3、切割-caspase-9和MMP-13的表达量均高于对照组、miR-27b-BMSC-exo组、BMSC-exo组(切割-caspase-3:P=0.025,P=0.020,P=0.006;切割-caspase-9:P=0.011,P=0.008,P=0.036;MMP-13:P=0.034,P=0.003,P=0.009);miR-27b-BMSC-exo组大鼠软骨细胞中切割-caspase-3、切割-caspase-9和MMP-13的表达量均低于BMSC-exo组(P=0.023,P=0.010,P=0.007)。(7)SD大鼠软骨缺损修复效果。直视下观察,miR-27b-BMSC-exo组SD大鼠软骨缺损修复效果优于对照组和BMSC-exo组。病理染色结果显示,对照组软骨层较薄,边缘不规则,软骨下骨和软骨无界限;miR-27b-BMSC-exo组和BMSC-exo组软骨层较厚,边缘光滑,软骨下骨和软骨界限清晰,且miR-27b-BMSC-exo组透明软骨修复效果优于BMSC-exo组。(8)软骨损伤修复标志蛋白表达的检测结果。免疫组织化学染色显示,对照组和BMSC-exo组SD大鼠软骨组织中MMP-13高表达,miR-27b-BMSC-exo组SD大鼠软骨组织中MMP-13低表达;对照组SD大鼠软骨组织中Ⅱ型胶原蛋白低表达,BMSC-exo组和miR-27b-BMSC-exo组SD大鼠软骨组织中Ⅱ型胶原蛋白高表达。MMP-13、Ⅱ型胶原蛋白表达阳性率的组间比较,差异有统计学意义(F=126.178,P=0.002;F=209.24,P=0.001)。miR-27b-BMSC-exo组SD大鼠软骨组织中Ⅱ型胶原蛋白表达量高于对照组和BMSC-exo组(P=0.005,P=0.012),MMP-13的表达量低于对照组和BMSC-exo组(P=0.029,P=0.014)。(9)SD大鼠软骨组织中软骨损伤相关蛋白表达的检测结果。切割-caspase-3、切割-caspase-9和MMP-13表达量的组间比较,差异有统计学意义(F=15.126,P=0.026;F=33.151,P=0.019;F=53.522,P=0.016),miR-27b-BMSC-exo组切割-caspase-3、切割-caspase-9和MMP-13的表达量均低于对照组和BMSC-exo组(切割-caspase-3:P=0.003,P=0.006;切割-caspase-9:P=0.001,P=0.019;MMP-13:P=0.007,P=0.008)。结论:miR-27b-BMSC-exo-chon-PLGA骨软骨复合体移植治疗软骨缺损,治疗效果显著,其作用机制可能与抑制caspase-3、caspase-9以及MMP-13的表达、促进Ⅱ型胶原蛋白的表达有关。

MicroRNA-27b-bone marrow mesenchymal stem cell derived exosomes-laden chondrocytes-poly(lactic-co-glycolic acid)osteochondral complex transplantation for treatment of osteochondral defects:an experimental study

Objective:To explore the curative effects and mechanisms of action of microRNA-27b(miR-27b)-bone marrow mesenchymal stem cell(BMSC)derived exosomes(exo)-laden chondrocytes(chon)-poly(lactic-co-glycolic acid)(PLGA)osteochondral complex transplantation for treatment of osteochondral defects.Methods:①The BMSC-derived exosomes(BMSC-exo)were prepared by using the cultured BMSC,and their morphologies were observed.The particle size and Zeta potential of BMSC-exo were measured,and the surface marker proteins CD9 and CD63 of BMSC-exo were detected by using Western Blot.②The 2nd and 3rd-generation BMSCs were divided into BMSC group and miR-27b-BMSC group.The BMSCs in BMSC group were cultured in RPMI-1640 medium without any intervention,while the BMSCs in miR-27b-BMSC group were infected with miR-27b-overexpressed recombinant adenovirus.After 24-hour cell cultivation,BMSC-exo and miRNA-27b-BMSC-exo were prepared;and the RNA were extracted from BMSC,BMSC-exo,miRNA-27b-BMSC and miRNA-27b-BMSC-exo respectively;and the expression of miR-27b was detected by using real-time quantitative PCR(RT-qPCR).③The miR-27b-BMSC-exo and chondrocytes of rats were stained with Dil and Hoechst33258 staining reagent respectively,and the uptake of miR-27b-BMSC-exo by chondrocytes of rats was observed under fluorescence microscope.④The rat chondrocytes were divided into BMSC-exo group and miR-27b-BMSC-exo group,and were inoculated into RPMI-1640 medium supplemented with BMSC-exo with final concentration of 80μg/mL and RPMI-1640 medium supplemented with miR-27b-BMSC-exo with final concentration of 80μg/mL respectively.The expression of miR-27b was detected by using RT-qPCR after 4-hour cell cultivation.⑤The rat chondrocytes were divided into control group,interleukin(IL)-1βgroup,miR-27b-BMSC-exo group and BMSC-exo group.The chondrocytes in control group were cultured in RPMI-1640 medium without any intervention,the chondrocytes in IL-1βgroup were cultured in RPMI-1640 medium supplemented with IL-1βwith final concentration of 10 ng/mL,the chondrocytes in miR-27b-BMSC-exo group were cultured in RPMI-1640 medium supplemented with IL-1βwith final concentration of 10 ng/mL and miR-27b-BMSC-exo with final concentration of 80μg/mL,and the chondrocytes in BMSC-exo group were cultured in RPMI-1640 medium supplemented with IL-1βwith final concentration of 10 ng/mL and BMSC-exo with final concentration of 80μg/mL.After cultured for continuous 0,1,2,3,4 and 8 hours,the well-growned rat chondrocytes were fetched out from each group respectively,and their cytoactive were detected by using MTT assay.After 24–hour cell cultivation,the total proteins of rat chondrocytes were extracted and the expressions of cartilage injury-related proteins including cysteine aspartic acid specific protease(caspase)-3,caspase-9 and matrix metalloproteinase(MMP)-13 were detected by using Western Blot method.⑥Eighteen 8-week-old female SD rats were obtained,and their left hind limb were fetched out.The 4.5-mm diameter and 1-mm depth cartilage defects on femoral trochlear grooves were created for buiding cartilage defect SD rat models.The 18 cartilage defect SD rat models were randomly divided into control group,miR-27b-BMSC-exo group and BMSC-exo group,and were implanted with chon-PLGA,miR-27b-BMSC-exo-chon-PLGA and BMSC-exo-chon-PLGA osteochondral complexes respectively.The SD rats were sacrificed after breeding for 12 weeks.The effects of osteochondral complexes in repairing cartilage defects were observed,and the expressions of typeⅡcollagen and MMP-13 were detected by using immunohistochemical staining.The total proteins were extracted from cartilage tissues of SD rats,and the expressions of caspase-3,caspase-9 and MMP-13 were detected by using Western Blot method.Results:①The disc-shaped vesicle-like membrane structure was found in BMSC-exo,and its particle size and Zeta potential was 92-115 nm and-22.13±2.1 mV respectively.The particle size of BMSC-exo with the highest proportion was 106 nm.The expression levels of CD9 and CD63 were significantly higher in BMSC-exo group compared to BMSC group(relative gray-scale value(GSV):6.513±0.714 vs 1.001±0.021,t=18.902,P=0.000;7.564±0.636 vs 1.026±0.027,t=25.158,P=0.000).②The expression levels of miR-27b in BMSC and exo were higher in miR-27b-BMSC group compared to BMSC group(relative expression level:46.785±8.153 vs 1.000±0.280,t=13.748,P=0.000;34.825±8.612 vs 1.000±0.325,t=9.621,P=0.000).③The rat chondrocytes were co-cultured with miR-27b-BMSC-exo for continuous 4 hours,and the miR-27b-BMSC-exo was taken by rat chondrocytes.④The expression level of miR-27b in rat chondrocytes was higher in miR-27b-BMSC-exo group compared to BMSC-exo group(relative expression level:3.315±0.523 vs 1.000±0.244,t=9.826,P=0.000).⑤There was interaction between time factor and group factor in rat chondrocytes activity(F=2.836,P=0.049).There was statistical difference in rat chondrocytes activity between the 4 groups in general,in other words,there was group effect(F=11.345,P=0.049).There was no statistical difference in rat chondrocytes activity between the 4 groups before the culture and after cultured for 1 and 2 hours(F=0.047,P=0.406;F=0.765,P=0.189;F=2.095,P=0.063).There was statistical difference in rat chondrocytes activity between the 4 groups after 3-,4-and 8-hour culture(F=4.720,P=0.039;F=7.421,P=0.021;F=95.348,P=0.000).The rat chondrocytes activity was lower in IL-1βgroup compared to control group,miR-27b-BMSC-exo group and BMSC-exo group(3 hours:P=0.002,P=0.011,P=0.026;4 hours:P=0.002,P=0.002,P=0.006;8 hours:P=0.004,P=0.004,P=0.011),and was higher in miR-27b-BMSC-exo group compared to control group and BMSC-exo group(3 hours:P=0.019,P=0.023;4 hours:P=0.015,P=0.002;8 hours:P=0.003,P=0.029).There was no statistical difference in rat chondrocytes activity between different timepoints,in other words,there was no time effect(F=0.258,P=0.083).⑥There was statistical difference in expression levels of cleaved-caspase-3,cleaved-caspase-9 and MMP-13 between the 4 groups(F=15.691,P=0.024;F=98.021,P=0.002;F=76.312,P=0.004).The expression levels of cleaved-caspase-3,cleaved-caspase-9 and MMP-13 in rat chondrocytes were higher in IL-1βgroup compared to control group,miR-27b-BMSC-exo group and BMSC-exo group(cleaved-caspase-3:P=0.025,P=0.020,P=0.006;cleaved-caspase-9:P=0.011,P=0.008,P=0.036;MMP-13:P=0.034,P=0.003,P=0.009),and were lower in miR-27b-BMSC-exo group compared to BMSC-exo group(P=0.023,P=0.010,P=0.007).⑦The cartilage defect remediation effectiveness of SD rats were better in miR-27b-BMSC-exo group compared to control group and BMSC-exo group under direct vision.The thin cartilage layer with irregular edge were found in rats of control group,and there was no boundary between subchondral bone and cartilage.While the thick cartilage layer with smooth edge were found in rats of miR-27b-BMSC-exo group and BMSC-exo group,and there was clear boundary between subchondral bone and cartilage,and the hyaline cartilage remediation effectiveness were better in miR-27b-BMSC-exo group compared to BMSC-exo group.⑧The results of immunohistochemical staining showed that the MMP-13 were high expressed in cartilage tissues of SD rats in control group and BMSC-exo group and low expressed in cartilage tissues of SD rats in miR-27b-BMSC-exo group,and the typeⅡcollagen were high expressed in cartilage tissues of SD rats in BMSC-exo group and miR-27b-BMSC-exo group and low expressed in cartilage tissues of SD rats in control group.There was statistical difference in positive rate of the expression of MMP-13 and typeⅡcollagen between the 3 groups(F=126.178,P=0.002;F=209.24,P=0.001).The expression level of typeⅡcollagen was higher and the expression level of MMP-13 was lower in chondrocytes of SD rats of miR-27b-BMSC-exo group compared to control group and BMSC-exo group(P=0.005,P=0.012;P=0.029,P=0.014).⑨There was statistical difference in expression levels of cleaved-caspase-3,cleaved-caspase-9 and MMP-13 between the 3 groups(F=15.126,P=0.026;F=33.151,P=0.019;F=53.522,P=0.016).The expression levels of cleaved-caspase-3,cleaved-caspase-9 and MMP-13 were lower in miR-27b-BMSC-exo group compared to control group and BMSC-exo group(cleaved-caspase-3:P=0.003,P=0.006;cleaved-caspase-9:P=0.001,P=0.019;MMP-13:P=0.007,P=0.008).Conclusion:The miR-27b-BMSC-exo-chon-PLGA osteochondral complex transplantation has significant clinical benefit in treatment of osteochondral defects,and its mechanisms of action may be that it can inhibit the expression of caspase-3,caspase-9 and MMP-13 and promote the expression of typeⅡcollagen.