hCDMP-2基因转染比格犬成肌细胞促进半月板纤维软骨组织损伤修复的研究
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摘要
随着现代经济的发展,创伤类型已由以开放性工种伤、暴力性殴打伤、钝挫伤为主转变为以交通伤、运动伤为主。关节损伤是交通伤、运动伤发病率最高的部位之一,相应地,关节内纤维软骨组织,如膝关节半月板、肩关节盂唇、髋关节盂唇、腕关节三角软骨盘等的损伤也日益增多,引起了创伤专家的极大关注。上述结构对相应关节有十分重要的功能,但损伤后自愈性差,如未得到及时治疗,最终将导致骨性关节炎,造成严重后果。近年采用组织工程与干细胞相结合的方法促进纤维软骨组织损伤的修复,但存在种子细胞的选择、获取与修复组织的质量(瘢痕愈合)等问题。成肌细胞来源丰富,位于体表容易获取,具有干细胞潜能,在体外生存能力强、繁殖快,被视为理想的供体细胞,在取材、操作及临床应用方面,优于其它干细胞(如骨髓间充质干细胞)。该细胞还具有耐缺血及促进损伤局部新生血管形成的作用,可能会解决纤维软骨组织损伤修复时局部血供不足的难题。软骨源性形态发生蛋白-2(CDMP-2)被证明能促进软骨修复,但不会发生骨化,还能促进纤维样组织的生成,是促进纤维软骨样组织损伤修复的理想因子。本研究以半月板为代表,拟将人源性CDMP-2(hCDMP-2)基因转染至体外分离纯化的比格犬成肌细胞中,以PLA/PGA支架为载体移植到损伤半月板局部,以期促进损伤局部达到纤维软骨样组织的修复,改善愈合质量,寻找促进该组织损伤修复的有效治疗途径。
目的:首先探索hCDMP-2细胞因子在体外诱导分离纯化的比格犬成肌细胞使其向纤维软骨细胞转化的可能性。如果结果显示诱导后细胞具有纤维软骨细胞表型,则通过设立相应的实验组与对照组,进一步探索比格犬成肌细胞在转染了hCDMP-2基因后对比格犬半月板纤维软骨组织损伤修复的作用。
材料与方法:取成年比格犬大腿后群肌肉,采用机械法与二步酶消化法溶解肌肉组织、扩增成肌细胞并采用差速贴壁与流式细胞仪分选相结合的方法纯化成肌细胞;对纯化后细胞进行形态学观察,绘制生长曲线,并采用流式细胞仪、RT-PCR及免疫细胞化学方法对所培养细胞进行成肌细胞表型及纯度的鉴定。在体外使用hCDMP-2细胞因子诱导培养纯化的比格犬成肌细胞,对诱导后细胞进行形态学观察,绘制生长曲线,并通过RT-PCR与免疫细胞化学方法检测成肌细胞在体外经过hCDMP-2因子诱导后是否具有纤维软骨细胞表型。在PubMed上检索hCDMP-2 CDS序列,进行CDS序列合成,hCDMP-2基因PCR产物的克隆,阳性克隆的筛选与鉴定,序列测定与BLAST分析。用慢病毒载体系统将hCDMP-2基因转染至体外分离纯化后的比格犬成肌细胞,采用实时荧光定量PCR(Real-time FluorescenceQuantitative PCR)方法进行转染成功率及转染后细胞hCDMP-2基因表达的鉴定,同时采用westernblot方法鉴定转染后细胞hCDMP-2蛋白的表达。使用进口PGA支架材料,通过特定的模具成形,滴加PLA,进行PLA/PGA支架的制作。制作比格犬半月板损伤模型:半月板前角距离前缘0.5cm处横贯整个半月板横径不保留基底部、宽2mm、关节囊缘厚2mm、关节腔缘厚1.5mm的缺损,损伤区涉及红-红区、红-白区与白-白区。进行比格犬半月板损伤修复体内分组动物实验:设立单纯缝合组(A组)、加有hCDMP-2细胞因子的PLA/PGA支架缝合组(B组)、单纯转染慢病毒的成肌细胞与PLA/PGA支架复合物移植缝合组(C组)以及转染hCDMP-2基因的成肌细胞与PLA/PGA支架复合物移植缝合组(D组)。分别在损伤修复后第3、8、12周取材,进行大体观察、形态学(HE染色、Safranin-O染色)及免疫组织化学(Ⅱ型胶原、S-100蛋白、Ⅰ型胶原)的定性检测,同时还分别对半月板不同缺损区(红-红区与白-白区)的修复组织进行组织学的定量检测(ELISA方法检测Ⅰ、Ⅱ型胶原蛋白的表达及Alcian Blue法检测GAG蛋白的表达)。
结果:
1、采用本课题方法分离纯化的比格犬成肌细胞成功率高,培养过程生长旺盛,存活率高。纯化后的P4代成肌细胞myogenin、MyoD基因呈阳性表达,肌细胞特异蛋白——desmin呈阳性表达,流式细胞仪检测时98%左右的细胞呈NCAM阳性表达,说明本法能够成功获得比格犬成肌细胞而且纯度高。
2、hCDMP-2因子在体外诱导培养纯化的P4代比格犬成肌细胞的结果显示:诱导后细胞形态从梭形的成肌细胞形态逐步转变成多边形与多角形的软骨细胞形态;RT-PCR检测显示,诱导后细胞在诱导后第20天(PI2代第11天)时开始表达软骨细胞特异的Sox9蛋白基因,之后随着时间的推移,诱导后细胞对该基因表达量也不断增加;免疫细胞化学方法检测显示,软骨细胞的另外两个标志性蛋白——Ⅱ型胶原与aggrecan及纤维软骨细胞的标志性蛋白——Ⅰ型胶原均在诱导后第25天(PI3代第5天)开始出现表达,而且阳性细胞率随着时间的延长而呈不断增加的趋势。以上结果可以初步证明体外分离培养并纯化的比格犬成肌细胞在hCDMP-2因子的诱导下能够向纤维软骨细胞表型转化,保证了我们进一步实验的可行性。
3、人工合成hCDMP-2 CDS全基因序列后进行质粒构建,然后通过慢病毒系统将其转染至体外分离纯化的比格犬成肌细胞。通过实时荧光定量PCR方法鉴定转染效率与转染后细胞hCDMP-2基因的表达,结果表明慢病毒转染hCDMP-2基因的成功率较高(约达71%),转染后细胞能表达hCDMP-2基因。Westernblot结果进一步表明转染后细胞可分泌hCDMP-2蛋白。
4、比格犬半月板损伤修复的体内动物实验结果显示:(1)在损伤修复后第3、8、12周,A组的半月板缺损修复区均未出现新生组织,缺损区的大小基本无变化。在损伤修复后第3周时,A、B、C组缺损修复区仍可见支架材料存在,支架材料体积较前缩小;到了第8周时,支架材料已完全消失,只留下与损伤时基本相同的缺损。以上结果表明:通过A组、B组与C组治疗的半月板缺损修复区未见有新生组织出现;本课题使用的PLA/PGA支架材料在体内会逐步降解,完全降解的时间为8周。(2)损伤修复后第3周时,D组缺损修复区可见支架材料存在,体积也较前缩小。分别取红-红区与白-白区的修复组织进行HE染色,结果显示:红-红区修复组织有少量软骨陷窝结构出现,而白-白区未有着色。分别取红-红区与白-白区的修复组织进行Safranin-O染色,结果显示:红-红区修复组织有部分着色,白-白区未有着色。取红-红区修复组织进行免疫组织化学检测,结果显示:Ⅱ型胶原、S-100蛋白、Ⅰ型胶原呈弱阳性表达。损伤修复后第8周时,可见D组缺损区的修复组织仍存在,基本充满整个缺损区,色泽较暗,质地较软,肉眼已看不见支架材料。分别取红-红区与白-白区的修复组织进行HE染色、Safranin-O染色与Ⅰ、Ⅱ型胶原、S-100蛋白的免疫组织化学检测,结果显示:HE染色见红-红区软骨陷窝结构增多,开始出现部分纤维结构,白-白区也出现了少量软骨陷窝结构,未见有纤维结构;Safranin-O染色见红-红区有着色,色泽及范围较前增加,白-白区也开始有部分着色;两个区的Ⅱ型胶原、S-100蛋白、Ⅰ型胶原的免疫组织化学检测均呈阳性。第12周时,见缺损修复区修复组织仍存在,体积大小基本无变化,色泽与周围正常半月板组织接近,质地较前变硬,致密度也明显增加。分别取红-红区与白-白区的修复组织进行HE染色、Safranin-O染色以及Ⅱ型胶原、S-100蛋白、Ⅰ型胶原的免疫组织化学检测,结果显示:HE染色见红-红区有明显软骨陷窝结构,周围纤维结构也较前明显,白-白区的软骨陷窝结构较前增多,未见有纤维结构;Safranin-O染色见两个区均有着色,色泽及范围均较前增加;免疫组织化学检测见两个区的Ⅱ型胶原、S-100蛋白、Ⅰ型胶原表达仍然均呈阳性。以上结果表明:D组采取的方法能够使本课题中的半月板缺损局部有新生修复组织出现,而且该修复组织具有纤维软骨组织的特征。(3)分别取D组第3、8、12周的红-红区与白-白区的修复组织(n=6),采用ELISA方法定量检测Ⅱ型与Ⅰ型胶原及Alcian Blue方法检测GAG蛋白的表达,结果显示:在损伤修复后第3周时,D组红-红区修复组织的Ⅱ型胶原、Ⅰ型胶原及GAG蛋白均有一定量的表达,而白-白区修复组织中的上述蛋白含量几乎为零;在损伤修复后第8周时,D组红-红区修复组织Ⅱ型胶原、Ⅰ型胶原及GAG蛋白含量较第3周时明显增加(P＜0.01),白-白区修复组织中的上述蛋白也出现表达;在损伤后第12周时D组红-红区与白-白区修复组织的Ⅱ型胶原、Ⅰ型胶原及GAG蛋白均有一定量的表达,两区各蛋白含量分别较第8周时明显增加(P＜0.01);D组在整个修复过程中,同一时间点的红-红区修复组织上述蛋白的含量明显多于白-白区(P＜0.01),但两区的上述蛋白含量均明显低于正常半月板组织的相应区域。以上结果除了进一步说明了D组采取的方法能够使本课题中的半月板缺损局部有类似于纤维软骨组织的新生修复组织出现,同时还说明半月板红-红区的损伤比白-白区修复得快。
结论:1、采用本课题方法分离的比格犬成肌细胞,存活率高、纯度高,说明本方法成功率高,简便易行,值得推广。2、经hCDMP-2因子在体外诱导后,纯化的比格犬成肌细胞能够向纤维软骨细胞表型转化。3、单纯缝合、单纯使用hCDMP-2因子与单纯的成肌细胞治疗在体内都不能促进本课题半月板缺损区纤维软骨组织损伤的修复。4、转染hCDMP-2基因的纯化成肌细胞可促进体内半月板纤维软骨组织各区损伤的修复,红-红区比白-白区修复得快。5、本研究中使用的方法为临床上治疗较大的半月板缺损提供了新的治疗思路。6、纯化的成肌细胞与hCDMP-2因子相结合对半月板纤维软骨组织损伤的修复有一定的促进作用,但相关治疗机制及将来的临床应用途径还需进一步探讨。
With the development of modern economy,the type of injury has transformed from battery injury and contusion to traffic accident injury and sports injury among which joint injury is of greatest morbidity. Accordingly,the injury of fibrocartilage in the joint,such as meniscus, glenoid labrun of shoulder and hip,triquetrous cartilage of wrist,became more and more often,which attracted the attention of specialists. Although the above structures are critical to corresponding joint,they cannot heal by themselves if injured and will result in severe consequence of osteoarthritis without prompt treatment.In recent years,tissue engineering combined with stem cell therapy was applied to treat the injury of fibrocartilage;however,there existed the problems of selection, acquisition of seed cells and the quality of repaired tissues.Myoblasts were regarded as ideal donor cells because of their abundant sources, obtainability,potentials of stem cells,strong survival ability and characteristics of rapid proliferation in vitro.Myoblasts were considered better in biopsy,manipulation and clinical application than other stem cells,such as bone mesenchymal stem cells.In addition,myoblasts have been proved to resist ischemia and promote neovascularization of injured tissue,which would be good to the repair of fibrocartilage with insufficient blood supply.Cartilage-derived morphogenic protein-2(CDMP-2)has been proved to promote cartilage repair without ossification and promote the fibro-tissue formation,thus CDMP-2 was considered an ideal cytokine for promoting the repair of fibrocartilage. Our research will transfect the human cartilage-derived morphogenic protein-2(hCDMP-2)gene into the isolated and purified canine myoblasts and transplant the complex of transfected myoblasts and PLA/PGA scaffold to the injured canine meniscus,by which we hope to promote the fibrocartilage-like repair of injured meniscus,improve the quality of healed tissue and look for an effective approach to treat the meniscal injury.
Objectives:To explore whether the isolated and purified canine myoblasts would be transformed to fibrochondrocytes in vitro when induced by hCDMP-2 cytokine.If succeeding,we will further explore the effects of canine myoblasts transfected with hCDMP-2 gene on the repair of canine meniscal fibrocartilage by setting up animal experiments with suitable grouped tests.
Materials and Methods:The muscle from the hindlimb of adult Beagle canine was obtained,from which myoblasts were isolated and harvested by mechanical decomposition and two-step enzyme digestion. Then the cells were purified by combination of method of differential adherent velocity and flow cytometry.The morphologic properities and the growth state of myoblasts were observed and the growth curves were drawn.In order to precisely characterize the phenotype and purity of these cells,flow cytometry,polymerase reaction with reverse transcription(RT-PCR)and immunocytochemistry analyses were performed.The purified canine myoblasts were induced by hCDMP-2 cytokine in vitro.The morphologic properities and the growth state of cells induced were observed and the growth curves were drawn.In order to precisely characterize the fibrochondrocyte-like phenotype of these cells,RT-PCR and immunocytochemistry analyses were performed. hCDMP-2 CDS sequence was retrieved in PubMed and the sequence was synthesized.The PCR production of hCDMP-2 gene was cloned,and then the positive clone was screened and identified by BLAST.The hCDMP-2 gene was then transfected into the purified canine myoblasts through lentivirus vector.The transfection ratio and expression of hCDMP-2 gene by transfected myoblasts was verified by Real-time Fluorescence Quantitative PCR.The expression of hCDMP-2 protein was also identified by westernblot.The PGA(Sigma,USA)scaffold material was shaped by specific mould and was added with PLA(Sigma, USA)to form the PLA/PGA scaffold.The model of canine meniscal injury was as follows:a defect of 2mm-width,2mm-thickness near the capsule,1.5mm-thickness near the the cavity,whole length of transverse diameter without reservation of basement at the anterior horn of the meniscus 0.5cm distant from the anterior edge was made,which involved the red-red,red-white and white-white areas of meniscus.To perform the animal experiments of canine meniscal repair in vivo,group of pure suture(group A),group of PLA/PGA scaffold containing hCDMP-2 cytokine combined with suture(group B),group of PLA/PGA scaffold containing myoblasts transfected by lentivirus combined with suture (group C)and group of PLA/PGA scaffold containing myoblasts transfected with hCDMP-2 gene combined with suture(group D)were established.The repaired tissues were taken off at 3~(rd),8~(th)and 12~(th)week after injury respectively and observed in gross appearance first.Then the morphology(HE staining and Safranin-O staining)was observed, immunocytochemistry analyses for collagenⅡ,S-100 and collagenⅠwere performed,quantative ELISA analyses(n=6)for collagenⅡand collagenⅠwere performed and quantative Alcian Blue analyses(n=6)for GAG were performed.
Results:1.Our method had a high possibility of success in obtaining the adult canine myoblasts with high survival rate in vitro and the myoblasts grew very well.Flow cytometry demonstrated that about 98% of P4(purified and passaged by 4 times)adult canine muscle cells were positive for CD56 and negative for fibroblastic marker TE7.RT-PCR analysis showed that P4 cells expressed the myogenic cell-related markers such as MyoD and Myogenin.Immunocytochemistry staining showed that P4 cells were positive for desmin.All these characteristics indicated successful primary culture of adult canine myoblasts with high purification.
2.After induced by hCDMP-2 cytokine in vitro,the morphology of the purified P4 canine myoblasts was transformed from spindle to polygon;the Sox9 gene which is specific for chondrocytes began to be expressed at the 20~(th)day after induction(the 11~(th)day of PI2)and the expression increased with time lapsing by RT-PCR;collagenⅡ,aggrecan which are also specific for chondrocytes and collagenⅠwhich is specific for fibrochondrocytes began to be expressed at the 25~(th)day after induction(the 5~(th)day of PI3)and the positive ratio increased with time lapsing by immunocytochemistry.The results above indicated that the purified canine myoblasts would have the phenotype of fibrochondrocytes when induced by hCDMP-2 cytokine in vitro,which ensure the feasibility of our further experiments.
3.The hCDMP-2 plasmid were successfully constructed with artificially synthesized CDS sequence and then the hCDMP-2 gene was transfected to the purified canine myoblasts through lentivirus vector in vitro with the transfection ratio of about 71%by Real-time Fluorescence Quantitative PCR.Myoblasts transfected with hCDMP-2 gene expressed hCDMP-2 gene by Real-time Fluorescence Quantitative PCR and hCDMP-2 protein by westernblot.
4.The animal experiments of canine meniscal repair in vivo showed: (1)There were no newborn tissue at the defective area of canine meniscus of group A at the 3~(rd),8~(th)and 12~(th)week after injury and the size of the area was nearly unchanged.At the 3~(rd)week after injury,the scaffold material still existed at the defective area with smaller size in group A,B and C. At the 8~(th)week,the scaffold material of group A,B and C disappeared completely and an identical defect was left.The above results showed that there was no newborn tissue in the canine meniscal defect by the treatment of group A,B and C and the PLA/PGA scaffold material could degrade with the degradation time of 8 weeks.(2)At the 3~(rd)week,the scaffold material still could be seen with smaller size at the defective area in group D.A little cartilage lacuna could be seen at the red-red area of repaired tissue by HE staining while the white-white area was not stained. The result of red-red area of repaired tissue stained by Safranin-O was positive while the white-white area was negtive.The collageⅡ,S-100 protein and collagenⅠwere weakly expressed at the red-red area of repaired tissue by immunohistochemistry.At the 8~(th)week,the repaired tissue in group D whose color and luster was dark and textile was soft still existed full of the defective area without scaffold material.More cartilage lacuna and a little fiber could be seen at the red-red area of repaired tissue by HE staining and a little cartilage lacuna without fiber could be seen at the white-white area.A larger scale of red-red area of repaired tissue with deeper color than at the 3~(rd)week was stained by Safranin-O staining and the white-white area was also stained with red corlor.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry.At the 12~(th)week,the repaired tissue in group D whose color and luster approximated to the normal meniscus,texitile was harder and density was higher still existed with size unchanged.Obvious cartilage lacuna and fiber could be seen at the red-red area of repaired tissue by HE staining and more cartilage lacuna still without fiber could be seen at the white-white area.Both the red-red area and the white-white area of repaired tissue were stained by Safranin-O staining with a larger scale and deeper color than at the 3~(rd)week.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry too.These results demonstrated that there were newborn tissues at the defective area by treatment of group D and the repaired tissue had the characteristics of fibrocartilage.(3)At the 3~(rd)week after injury,some collageⅡ,collagenⅠand GAG were detected at the red-red area of repaired tissue in group D while they were not detected at the white-white area.At the 8~(th)week,the content of collageⅡ,collagenⅠand GAG at red-red area of repaired tissue in group D were higher than at the 3~(rd)week(P＜0.01)and these proteins were also detected at the white-white area.At the 12~(th)week, collageⅡ,collagenⅠand GAG with higher contents than at 8~(th)week (P＜0.01)were detected both at the red-red area and at the white-white area of repaired tissue in group D.At the same time during the whole process of repair in group D,the content of these proteins at the red-red area of repaired tissue was significantly higher than at the white-white area(P＜0.01),which indicated that the repair speed at the red-red area of repaired tissue was higher than that at the white-white area.However,at the same time during the whole process of repair in group D,the content of these proteins at the red-red and the white-white area was significantly lower than that of at respective area of normal meniscus(P＜0.01).
Conclusions:1.Our method for isolating and purifying myoblasts in vitro proved to be convenient and practical with high possibility of success and low price.Studies of cell therapy using primary myoblasts can now be broadly applied to canine models of human muscle and non-muscle diseases.2.The purified canine myoblasts could have the phenotype of fibrochondrocytes after induced by hCDMP-2 cytokine.3. The canine meniscal defect in our research could not be repaired by pure suture,applying hCDMP-2 cytokine only or using purified myoblast therapy only.4.The purified myoblasts transfected with hCDMP-2 gene could promote the repair of canine meniscal fibrocartilage of each area and the repair speed of the red-red area was higher than that of the white-white area.5.The method used in our research may provide a new approach to treat large meniscal defects.6.The therapy of purified myoblasts combined with hCDMP-2 cytokine could promote the the repair of canine meniscal fibrocartilage,however related therapeutic mechanism and approach of future clinical application remains unknown.
目的:首先探索hCDMP-2细胞因子在体外诱导分离纯化的比格犬成肌细胞使其向纤维软骨细胞转化的可能性。如果结果显示诱导后细胞具有纤维软骨细胞表型,则通过设立相应的实验组与对照组,进一步探索比格犬成肌细胞在转染了hCDMP-2基因后对比格犬半月板纤维软骨组织损伤修复的作用。
材料与方法:取成年比格犬大腿后群肌肉,采用机械法与二步酶消化法溶解肌肉组织、扩增成肌细胞并采用差速贴壁与流式细胞仪分选相结合的方法纯化成肌细胞;对纯化后细胞进行形态学观察,绘制生长曲线,并采用流式细胞仪、RT-PCR及免疫细胞化学方法对所培养细胞进行成肌细胞表型及纯度的鉴定。在体外使用hCDMP-2细胞因子诱导培养纯化的比格犬成肌细胞,对诱导后细胞进行形态学观察,绘制生长曲线,并通过RT-PCR与免疫细胞化学方法检测成肌细胞在体外经过hCDMP-2因子诱导后是否具有纤维软骨细胞表型。在PubMed上检索hCDMP-2 CDS序列,进行CDS序列合成,hCDMP-2基因PCR产物的克隆,阳性克隆的筛选与鉴定,序列测定与BLAST分析。用慢病毒载体系统将hCDMP-2基因转染至体外分离纯化后的比格犬成肌细胞,采用实时荧光定量PCR(Real-time FluorescenceQuantitative PCR)方法进行转染成功率及转染后细胞hCDMP-2基因表达的鉴定,同时采用westernblot方法鉴定转染后细胞hCDMP-2蛋白的表达。使用进口PGA支架材料,通过特定的模具成形,滴加PLA,进行PLA/PGA支架的制作。制作比格犬半月板损伤模型:半月板前角距离前缘0.5cm处横贯整个半月板横径不保留基底部、宽2mm、关节囊缘厚2mm、关节腔缘厚1.5mm的缺损,损伤区涉及红-红区、红-白区与白-白区。进行比格犬半月板损伤修复体内分组动物实验:设立单纯缝合组(A组)、加有hCDMP-2细胞因子的PLA/PGA支架缝合组(B组)、单纯转染慢病毒的成肌细胞与PLA/PGA支架复合物移植缝合组(C组)以及转染hCDMP-2基因的成肌细胞与PLA/PGA支架复合物移植缝合组(D组)。分别在损伤修复后第3、8、12周取材,进行大体观察、形态学(HE染色、Safranin-O染色)及免疫组织化学(Ⅱ型胶原、S-100蛋白、Ⅰ型胶原)的定性检测,同时还分别对半月板不同缺损区(红-红区与白-白区)的修复组织进行组织学的定量检测(ELISA方法检测Ⅰ、Ⅱ型胶原蛋白的表达及Alcian Blue法检测GAG蛋白的表达)。
结果:
1、采用本课题方法分离纯化的比格犬成肌细胞成功率高,培养过程生长旺盛,存活率高。纯化后的P4代成肌细胞myogenin、MyoD基因呈阳性表达,肌细胞特异蛋白——desmin呈阳性表达,流式细胞仪检测时98%左右的细胞呈NCAM阳性表达,说明本法能够成功获得比格犬成肌细胞而且纯度高。
2、hCDMP-2因子在体外诱导培养纯化的P4代比格犬成肌细胞的结果显示:诱导后细胞形态从梭形的成肌细胞形态逐步转变成多边形与多角形的软骨细胞形态;RT-PCR检测显示,诱导后细胞在诱导后第20天(PI2代第11天)时开始表达软骨细胞特异的Sox9蛋白基因,之后随着时间的推移,诱导后细胞对该基因表达量也不断增加;免疫细胞化学方法检测显示,软骨细胞的另外两个标志性蛋白——Ⅱ型胶原与aggrecan及纤维软骨细胞的标志性蛋白——Ⅰ型胶原均在诱导后第25天(PI3代第5天)开始出现表达,而且阳性细胞率随着时间的延长而呈不断增加的趋势。以上结果可以初步证明体外分离培养并纯化的比格犬成肌细胞在hCDMP-2因子的诱导下能够向纤维软骨细胞表型转化,保证了我们进一步实验的可行性。
3、人工合成hCDMP-2 CDS全基因序列后进行质粒构建,然后通过慢病毒系统将其转染至体外分离纯化的比格犬成肌细胞。通过实时荧光定量PCR方法鉴定转染效率与转染后细胞hCDMP-2基因的表达,结果表明慢病毒转染hCDMP-2基因的成功率较高(约达71%),转染后细胞能表达hCDMP-2基因。Westernblot结果进一步表明转染后细胞可分泌hCDMP-2蛋白。
4、比格犬半月板损伤修复的体内动物实验结果显示:(1)在损伤修复后第3、8、12周,A组的半月板缺损修复区均未出现新生组织,缺损区的大小基本无变化。在损伤修复后第3周时,A、B、C组缺损修复区仍可见支架材料存在,支架材料体积较前缩小;到了第8周时,支架材料已完全消失,只留下与损伤时基本相同的缺损。以上结果表明:通过A组、B组与C组治疗的半月板缺损修复区未见有新生组织出现;本课题使用的PLA/PGA支架材料在体内会逐步降解,完全降解的时间为8周。(2)损伤修复后第3周时,D组缺损修复区可见支架材料存在,体积也较前缩小。分别取红-红区与白-白区的修复组织进行HE染色,结果显示:红-红区修复组织有少量软骨陷窝结构出现,而白-白区未有着色。分别取红-红区与白-白区的修复组织进行Safranin-O染色,结果显示:红-红区修复组织有部分着色,白-白区未有着色。取红-红区修复组织进行免疫组织化学检测,结果显示:Ⅱ型胶原、S-100蛋白、Ⅰ型胶原呈弱阳性表达。损伤修复后第8周时,可见D组缺损区的修复组织仍存在,基本充满整个缺损区,色泽较暗,质地较软,肉眼已看不见支架材料。分别取红-红区与白-白区的修复组织进行HE染色、Safranin-O染色与Ⅰ、Ⅱ型胶原、S-100蛋白的免疫组织化学检测,结果显示:HE染色见红-红区软骨陷窝结构增多,开始出现部分纤维结构,白-白区也出现了少量软骨陷窝结构,未见有纤维结构;Safranin-O染色见红-红区有着色,色泽及范围较前增加,白-白区也开始有部分着色;两个区的Ⅱ型胶原、S-100蛋白、Ⅰ型胶原的免疫组织化学检测均呈阳性。第12周时,见缺损修复区修复组织仍存在,体积大小基本无变化,色泽与周围正常半月板组织接近,质地较前变硬,致密度也明显增加。分别取红-红区与白-白区的修复组织进行HE染色、Safranin-O染色以及Ⅱ型胶原、S-100蛋白、Ⅰ型胶原的免疫组织化学检测,结果显示:HE染色见红-红区有明显软骨陷窝结构,周围纤维结构也较前明显,白-白区的软骨陷窝结构较前增多,未见有纤维结构;Safranin-O染色见两个区均有着色,色泽及范围均较前增加;免疫组织化学检测见两个区的Ⅱ型胶原、S-100蛋白、Ⅰ型胶原表达仍然均呈阳性。以上结果表明:D组采取的方法能够使本课题中的半月板缺损局部有新生修复组织出现,而且该修复组织具有纤维软骨组织的特征。(3)分别取D组第3、8、12周的红-红区与白-白区的修复组织(n=6),采用ELISA方法定量检测Ⅱ型与Ⅰ型胶原及Alcian Blue方法检测GAG蛋白的表达,结果显示:在损伤修复后第3周时,D组红-红区修复组织的Ⅱ型胶原、Ⅰ型胶原及GAG蛋白均有一定量的表达,而白-白区修复组织中的上述蛋白含量几乎为零;在损伤修复后第8周时,D组红-红区修复组织Ⅱ型胶原、Ⅰ型胶原及GAG蛋白含量较第3周时明显增加(P＜0.01),白-白区修复组织中的上述蛋白也出现表达;在损伤后第12周时D组红-红区与白-白区修复组织的Ⅱ型胶原、Ⅰ型胶原及GAG蛋白均有一定量的表达,两区各蛋白含量分别较第8周时明显增加(P＜0.01);D组在整个修复过程中,同一时间点的红-红区修复组织上述蛋白的含量明显多于白-白区(P＜0.01),但两区的上述蛋白含量均明显低于正常半月板组织的相应区域。以上结果除了进一步说明了D组采取的方法能够使本课题中的半月板缺损局部有类似于纤维软骨组织的新生修复组织出现,同时还说明半月板红-红区的损伤比白-白区修复得快。
结论:1、采用本课题方法分离的比格犬成肌细胞,存活率高、纯度高,说明本方法成功率高,简便易行,值得推广。2、经hCDMP-2因子在体外诱导后,纯化的比格犬成肌细胞能够向纤维软骨细胞表型转化。3、单纯缝合、单纯使用hCDMP-2因子与单纯的成肌细胞治疗在体内都不能促进本课题半月板缺损区纤维软骨组织损伤的修复。4、转染hCDMP-2基因的纯化成肌细胞可促进体内半月板纤维软骨组织各区损伤的修复,红-红区比白-白区修复得快。5、本研究中使用的方法为临床上治疗较大的半月板缺损提供了新的治疗思路。6、纯化的成肌细胞与hCDMP-2因子相结合对半月板纤维软骨组织损伤的修复有一定的促进作用,但相关治疗机制及将来的临床应用途径还需进一步探讨。
With the development of modern economy,the type of injury has transformed from battery injury and contusion to traffic accident injury and sports injury among which joint injury is of greatest morbidity. Accordingly,the injury of fibrocartilage in the joint,such as meniscus, glenoid labrun of shoulder and hip,triquetrous cartilage of wrist,became more and more often,which attracted the attention of specialists. Although the above structures are critical to corresponding joint,they cannot heal by themselves if injured and will result in severe consequence of osteoarthritis without prompt treatment.In recent years,tissue engineering combined with stem cell therapy was applied to treat the injury of fibrocartilage;however,there existed the problems of selection, acquisition of seed cells and the quality of repaired tissues.Myoblasts were regarded as ideal donor cells because of their abundant sources, obtainability,potentials of stem cells,strong survival ability and characteristics of rapid proliferation in vitro.Myoblasts were considered better in biopsy,manipulation and clinical application than other stem cells,such as bone mesenchymal stem cells.In addition,myoblasts have been proved to resist ischemia and promote neovascularization of injured tissue,which would be good to the repair of fibrocartilage with insufficient blood supply.Cartilage-derived morphogenic protein-2(CDMP-2)has been proved to promote cartilage repair without ossification and promote the fibro-tissue formation,thus CDMP-2 was considered an ideal cytokine for promoting the repair of fibrocartilage. Our research will transfect the human cartilage-derived morphogenic protein-2(hCDMP-2)gene into the isolated and purified canine myoblasts and transplant the complex of transfected myoblasts and PLA/PGA scaffold to the injured canine meniscus,by which we hope to promote the fibrocartilage-like repair of injured meniscus,improve the quality of healed tissue and look for an effective approach to treat the meniscal injury.
Objectives:To explore whether the isolated and purified canine myoblasts would be transformed to fibrochondrocytes in vitro when induced by hCDMP-2 cytokine.If succeeding,we will further explore the effects of canine myoblasts transfected with hCDMP-2 gene on the repair of canine meniscal fibrocartilage by setting up animal experiments with suitable grouped tests.
Materials and Methods:The muscle from the hindlimb of adult Beagle canine was obtained,from which myoblasts were isolated and harvested by mechanical decomposition and two-step enzyme digestion. Then the cells were purified by combination of method of differential adherent velocity and flow cytometry.The morphologic properities and the growth state of myoblasts were observed and the growth curves were drawn.In order to precisely characterize the phenotype and purity of these cells,flow cytometry,polymerase reaction with reverse transcription(RT-PCR)and immunocytochemistry analyses were performed.The purified canine myoblasts were induced by hCDMP-2 cytokine in vitro.The morphologic properities and the growth state of cells induced were observed and the growth curves were drawn.In order to precisely characterize the fibrochondrocyte-like phenotype of these cells,RT-PCR and immunocytochemistry analyses were performed. hCDMP-2 CDS sequence was retrieved in PubMed and the sequence was synthesized.The PCR production of hCDMP-2 gene was cloned,and then the positive clone was screened and identified by BLAST.The hCDMP-2 gene was then transfected into the purified canine myoblasts through lentivirus vector.The transfection ratio and expression of hCDMP-2 gene by transfected myoblasts was verified by Real-time Fluorescence Quantitative PCR.The expression of hCDMP-2 protein was also identified by westernblot.The PGA(Sigma,USA)scaffold material was shaped by specific mould and was added with PLA(Sigma, USA)to form the PLA/PGA scaffold.The model of canine meniscal injury was as follows:a defect of 2mm-width,2mm-thickness near the capsule,1.5mm-thickness near the the cavity,whole length of transverse diameter without reservation of basement at the anterior horn of the meniscus 0.5cm distant from the anterior edge was made,which involved the red-red,red-white and white-white areas of meniscus.To perform the animal experiments of canine meniscal repair in vivo,group of pure suture(group A),group of PLA/PGA scaffold containing hCDMP-2 cytokine combined with suture(group B),group of PLA/PGA scaffold containing myoblasts transfected by lentivirus combined with suture (group C)and group of PLA/PGA scaffold containing myoblasts transfected with hCDMP-2 gene combined with suture(group D)were established.The repaired tissues were taken off at 3~(rd),8~(th)and 12~(th)week after injury respectively and observed in gross appearance first.Then the morphology(HE staining and Safranin-O staining)was observed, immunocytochemistry analyses for collagenⅡ,S-100 and collagenⅠwere performed,quantative ELISA analyses(n=6)for collagenⅡand collagenⅠwere performed and quantative Alcian Blue analyses(n=6)for GAG were performed.
Results:1.Our method had a high possibility of success in obtaining the adult canine myoblasts with high survival rate in vitro and the myoblasts grew very well.Flow cytometry demonstrated that about 98% of P4(purified and passaged by 4 times)adult canine muscle cells were positive for CD56 and negative for fibroblastic marker TE7.RT-PCR analysis showed that P4 cells expressed the myogenic cell-related markers such as MyoD and Myogenin.Immunocytochemistry staining showed that P4 cells were positive for desmin.All these characteristics indicated successful primary culture of adult canine myoblasts with high purification.
2.After induced by hCDMP-2 cytokine in vitro,the morphology of the purified P4 canine myoblasts was transformed from spindle to polygon;the Sox9 gene which is specific for chondrocytes began to be expressed at the 20~(th)day after induction(the 11~(th)day of PI2)and the expression increased with time lapsing by RT-PCR;collagenⅡ,aggrecan which are also specific for chondrocytes and collagenⅠwhich is specific for fibrochondrocytes began to be expressed at the 25~(th)day after induction(the 5~(th)day of PI3)and the positive ratio increased with time lapsing by immunocytochemistry.The results above indicated that the purified canine myoblasts would have the phenotype of fibrochondrocytes when induced by hCDMP-2 cytokine in vitro,which ensure the feasibility of our further experiments.
3.The hCDMP-2 plasmid were successfully constructed with artificially synthesized CDS sequence and then the hCDMP-2 gene was transfected to the purified canine myoblasts through lentivirus vector in vitro with the transfection ratio of about 71%by Real-time Fluorescence Quantitative PCR.Myoblasts transfected with hCDMP-2 gene expressed hCDMP-2 gene by Real-time Fluorescence Quantitative PCR and hCDMP-2 protein by westernblot.
4.The animal experiments of canine meniscal repair in vivo showed: (1)There were no newborn tissue at the defective area of canine meniscus of group A at the 3~(rd),8~(th)and 12~(th)week after injury and the size of the area was nearly unchanged.At the 3~(rd)week after injury,the scaffold material still existed at the defective area with smaller size in group A,B and C. At the 8~(th)week,the scaffold material of group A,B and C disappeared completely and an identical defect was left.The above results showed that there was no newborn tissue in the canine meniscal defect by the treatment of group A,B and C and the PLA/PGA scaffold material could degrade with the degradation time of 8 weeks.(2)At the 3~(rd)week,the scaffold material still could be seen with smaller size at the defective area in group D.A little cartilage lacuna could be seen at the red-red area of repaired tissue by HE staining while the white-white area was not stained. The result of red-red area of repaired tissue stained by Safranin-O was positive while the white-white area was negtive.The collageⅡ,S-100 protein and collagenⅠwere weakly expressed at the red-red area of repaired tissue by immunohistochemistry.At the 8~(th)week,the repaired tissue in group D whose color and luster was dark and textile was soft still existed full of the defective area without scaffold material.More cartilage lacuna and a little fiber could be seen at the red-red area of repaired tissue by HE staining and a little cartilage lacuna without fiber could be seen at the white-white area.A larger scale of red-red area of repaired tissue with deeper color than at the 3~(rd)week was stained by Safranin-O staining and the white-white area was also stained with red corlor.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry.At the 12~(th)week,the repaired tissue in group D whose color and luster approximated to the normal meniscus,texitile was harder and density was higher still existed with size unchanged.Obvious cartilage lacuna and fiber could be seen at the red-red area of repaired tissue by HE staining and more cartilage lacuna still without fiber could be seen at the white-white area.Both the red-red area and the white-white area of repaired tissue were stained by Safranin-O staining with a larger scale and deeper color than at the 3~(rd)week.The collageⅡ,S-100 protein and collagenⅠwere all expressed at both the red-red area and white-white area of repaired tissue by immunohistochemistry too.These results demonstrated that there were newborn tissues at the defective area by treatment of group D and the repaired tissue had the characteristics of fibrocartilage.(3)At the 3~(rd)week after injury,some collageⅡ,collagenⅠand GAG were detected at the red-red area of repaired tissue in group D while they were not detected at the white-white area.At the 8~(th)week,the content of collageⅡ,collagenⅠand GAG at red-red area of repaired tissue in group D were higher than at the 3~(rd)week(P＜0.01)and these proteins were also detected at the white-white area.At the 12~(th)week, collageⅡ,collagenⅠand GAG with higher contents than at 8~(th)week (P＜0.01)were detected both at the red-red area and at the white-white area of repaired tissue in group D.At the same time during the whole process of repair in group D,the content of these proteins at the red-red area of repaired tissue was significantly higher than at the white-white area(P＜0.01),which indicated that the repair speed at the red-red area of repaired tissue was higher than that at the white-white area.However,at the same time during the whole process of repair in group D,the content of these proteins at the red-red and the white-white area was significantly lower than that of at respective area of normal meniscus(P＜0.01).
Conclusions:1.Our method for isolating and purifying myoblasts in vitro proved to be convenient and practical with high possibility of success and low price.Studies of cell therapy using primary myoblasts can now be broadly applied to canine models of human muscle and non-muscle diseases.2.The purified canine myoblasts could have the phenotype of fibrochondrocytes after induced by hCDMP-2 cytokine.3. The canine meniscal defect in our research could not be repaired by pure suture,applying hCDMP-2 cytokine only or using purified myoblast therapy only.4.The purified myoblasts transfected with hCDMP-2 gene could promote the repair of canine meniscal fibrocartilage of each area and the repair speed of the red-red area was higher than that of the white-white area.5.The method used in our research may provide a new approach to treat large meniscal defects.6.The therapy of purified myoblasts combined with hCDMP-2 cytokine could promote the the repair of canine meniscal fibrocartilage,however related therapeutic mechanism and approach of future clinical application remains unknown.
引文
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3.Szoeke CE,Cicuttini FM,Guthrie JR,et al.Factors affecting the prevalence of osteoarthritis in healthy middle-aged women:Data from the longitudinal Melbourne Women's Midlife Health Project.Bone.2006,39(5):1149-55.
4.Hennerbichler A,Moutos FT,Hennerbichler D,et al.Repair response of the inner and outer regions of the porcine meniscus in vitro.Am J Sports Med.,2007,35(5):754-62.
5. King D The healing of the semilunar cartilages. J Bone Joint Surg Am. 1936, 18:333-342.
6. Webber RJ, York JL, Vanderschilden JL, et al. An organ culture model for assaying wound repair of the fibrocartilaginous knee joint meniscus. Am J Sports Med, 1989, 17(3):393-400.
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