基因修饰BMSCs抑制增生性瘢痕的实验研究

目的 通过将转染TGF-β3c2s2基因的兔BMSCs移植于兔耳瘢痕模型,观察转基因BMSCs对创面愈合后增生性瘢痕形成的影响. 方法 成年健康日本大耳白兔20只,体重1.7～2.5 kg,雌雄不拘.取第3代兔BMSCs,经Ad-TGF-β3c2s2在感染复数150下转染,培养孵育24 h后,调整细胞浓度为1×105/mL备用.将纯化、浓缩的Ad-TGF-β3c2s2颗粒,DMEM/F12(不含FBS)稀释为1×108pfu/mL备用.于20只日本大耳白兔双侧兔耳分别制备两个2 cm×2 cm大小的腹侧全层皮肤、软骨缺损创面.将每只兔的4个创面随机分为空白对照组(A组)、Ad-TGF-β3c2s2组(B组)、BMSCs组(C组)和BMSCs/Ad-TGF-β3c2s2组(D组),并以自身正常皮肤作为正常对照(E组).将备好的细胞及病毒液分别按创面分组进行局部移植.于术后21、45、90 d行大体观察、瘢痕硬度和厚度测定、HE染色和免疫组织化学检测. 结果 大体观察A、B、C组上皮化后创面均逐渐形成不同程度的瘢痕,伤后45 d瘢痕增生程度达高峰,明显高出皮肤表面,持续至90 d,D组在观察期内均无明显高出周围皮肤的瘢痕形成.术后45 d和90 d,A、B、C组瘢痕厚度和硬度明显高于D组,且差异均有统计学意义(P＜0.01),而B组低于A、C组,差异有统计学意义(P＜0.01);D组愈合后创面厚度和硬度与正常皮肤接近.HE染色观察A、C组伤后45 d可见浅层组织结构排列紊乱,胶原纤维粗大,呈交错网织状排列;B组和D组结构与正常皮肤接近,但较E组胶原排列致密,表皮较A、C组薄;90 d各组结构与45 d相似.BrdU免疫组织化学观察,伤后21、45 d,C、D组均能见到散在分布、胞核染色阳性的棕色细胞,A、B、E组均为阴性. 结论 创面局部移植人TGF-β3c2s2基因修饰的BMSCs,具有抑制创面愈合后增生性瘢痕的作用.

EXPERIMENTAL STUDY ON BMSCs TRANSFECTED BY ENDOGENE INHIBITING HYPERTROPHIC SCAR

OBJECTIVE: To investigate the effects on forming of hypertrophic scar after BMSCs infected with adenovirus carrying TGF-beta3c2s2 were transplanted into the wound of animal scar model. METHODS: The third passage of rabbit's BMSCs were infected with 150 mutiple infection, and were cultured 24 hours. The concentration of the BMSCs infected with recombinant adenovirus containing the TGF-beta3c2s2 gene was 1 x 10(5) cell/mL. The purified and evaporated recombinant adenovirus grains containing the TGF-beta3c2s2 gene were diluted by DMEM/F12 (without FBS) to 1 x 10(8) pfu/mL. The animal scar model of the standard Japanese big ear rabbit was established. Eighty wounds were generated on the gastroside of ear and were randomized to 4 groups in each rabbit, which were divided into 3 control groups (A: control, B: Ad-TGF-beta3c2s2, C: BMSCs) and 1 experimental group (D: BMSCs/Ad-TGF-beta3c2s2). Then the wounds were tranplanted with cells. On 45 days and 90 days after wounded, thickness and hardness of scars were measured with color ultrasound diagnostic unit and especial measurement for skin and scar hardness. On 21, 45 and 90 days, three specimens were harvested respectively for further histological study. RESULTS: The wound of groups A, B, C gradually formed the different degree scars after epithelialization. The hyperplasty of scars reached peak on 45 days after wounded and lasted about 90 days. There was no prominent scar formed in group D during the whole observed procedure. Thickness and hardness of scar of group D and group E were approximate on 45 days and 90 days. Thickness and hardness of scar of groups A, B and C were lower than those of group D (P < 0.01), and group B showed more lower than group A and group C (P < 0.01). Disorder structure and overlapping arrangement, enlargement collagen fibers were showed in the HE histological sections of the scars of groups A, C. The structure of the scars of groups B, C were similar to Group E. The constitutions of groups A, B, C, D on 90 days resembled to each one on 45 days. In section of immunohistochemistry after wounded on 21 days and 45 days, positive expressions of BrdU in nucleus of Groups C, D were observed. Negative expressions of BrdU in Groups A, B, E were showed. CONCLUSION: BMSCs with Ad-TGFbeta3c2s2 gene transplanted into wound could inhibit the forming of hypertrophic scar.