CollagenⅤ在推拿促进长期大负荷运动大鼠肌肉干细胞自我更新中的作用研究

目的:观察推拿对长期大负荷运动大鼠骨骼肌损伤修复、后肢抓力及CollagenⅤ表达的影响,探讨推拿促进大鼠肌肉干细胞自我更新的机制。方法:32只雄性SD大鼠随机分为空白对照组(C组)、长期大负荷运动组(E组)、长期大负荷运动+推拿组(ET组)、长期大负荷运动+假推拿组(ES组)。C组不做任何处理,E组、ET组和ES组进行4周大负荷跑台运动制备长期大负荷运动损伤模型;E组不予任何干预;ET组于每次运动后6h予以推拿干预;ES组于每次运动后6h仅予以抚摸干预。测定初始及4周后大鼠的体重和后肢抓力。末次干预后24h取大鼠两侧腓肠肌,采用透射电镜观察肌纤维超微结构变化;免疫组织化学染色检测CollagenⅤ表达水平;实时荧光定量PCR检测Col5a1和Col5a3 mRNA表达水平;Western Blot检测Pax7、MyoD和Myf5蛋白表达水平;双重免疫荧光染色检测Pax7与CollagenⅤ共定位。结果:与C组相比,E组、ES组和ET组大鼠后肢抓力增加值差异显著(P<0.01),E组和ES组超微结构均出现肌原纤维断裂等变化,E组和ES组CollagenⅤ、Pax7、MyoD、Myf5、Col5a1和Col5a3 mRNA以及Pax7与CollagenⅤ共定位表达显著降低(P<0.05);与E组相比,ET组后肢抓力增加值差异显著(P<0.05),超微结构表现良好,CollagenⅤ、Pax7、MyoD、Myf5、Col5a3 mRNA以及Pax7与CollagenⅤ共定位表达显著升高(P<0.05)。结论:推拿可通过上调CollagenⅤ表达,促进大鼠骨骼肌肌肉干细胞自我更新,从而加速修复长期大负荷运动诱发的超微结构损伤,增强肌肉力量。

Effects of angiotensin Ⅱ receptor antagonist on expression of collagen Ⅲ,collagen Ⅴ,and transforming growth factor β_1 in the airway walls of sensitized rats

Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling,the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma,we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ,collagen Ⅴ,and transforming growth factor β_1 (TGF-β_1) mRNA and protein in the airway walls of sensitized rats.Methods Forty Wistar rats were randomly divided into 5 groups: control group,sensitized group,and valsartan groups 1,2,and 3. The rats in the sensitized group and in valsartan groups 1,2,and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1,2,and 3 were drenched with valsartan (10 μg, 20 μg,or 30 μg,respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ,collagen Ⅴ,and TGF-β_1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β_1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen Ⅴ was significantly higher in rats from the sensitized group (7.73±0.81, 1.34±0.28) and from valsartan groups 1,2,and 3 (5.73±0.64, 1.13±0.15; 4.96±0.51, 0.98±0.08; 4.43±0.35, 0.93±0.06,respectively) than those in the control group (2.65±0.38, 0.67±0.08,P <0.05). In addition,collagen levels were significantly lower in valsartan groups 1,2,and 3 than those from the sensitized group ( P <0.05). The expression of TGF-β_1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49%±3.46%,29.73%±3.25%) and from valsartan groups 1,2,and 3 (16.47%±1.94%, 19.41%±1.87%; 14.38%±1.58%, 18.29%±1.43%; 12.96%±1.73%, 18.63%±1.11%,respectively) than that from the control group (7.84%±1.61%, 5.63%±1.07%,P <0.05). TGF-β_1 mRNA and protein levels were significantly lower in valsartan groups 1,2,and 3 than that in the sensitized group ( P <0.05). Conclusions Angiotensin Ⅱ receptor antagonist valsartan can suppress synthesis of collagen Ⅲ and collagen Ⅴ by downregulating TGF-β_1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy.

Ⅰ型和Ⅴ型胶原蛋白通过YAP核转位调节大鼠胰岛β细胞增殖的研究

目的探讨Yes相关蛋白(YAP)核转位对Ⅰ型和Ⅴ型胶原蛋白包被的大鼠胰岛β细胞(INS-1细胞)增殖的影响。方法将INS-1细胞分为空白组(未包被胶原蛋白,正常培养)、对照组(包被40.00 mg·mL^(-1)的Ⅰ型胶原蛋白)和实验组(包被5.00 mg·mL^(-1)的Ⅴ型胶原蛋白),并培养48 h。用噻唑蓝法检测细胞的存活率,用PI流式细胞术检测细胞周期,用蛋白质印迹法检测YAP蛋白的表达情况。结果实验组、对照组和空白组的干预48 h后细胞存活率分别为(46.65±4.29)%、(112.90±3.20)%和(100.00±0.00)%,S+G2/M期比例分别为(30.51±1.05)%、(41.59±0.90)%和(36.15±1.08)%,细胞核中YAP相对表达水平分别为0.78±0.06、1.17±0.04和1.00±0.00,细胞质中YAP相对表达水平分别为1.15±0.02、0.78±0.05和1.00±0.00,实验组和对照组的上述指标与空白组比较,差异均有统计学意义(均P<0.05)。结论Ⅰ型胶原蛋白包被培养促进YAP核转位,从而促进INS-1细胞增殖;而Ⅴ型胶原蛋白包被培养抑制YAP核转位,从而抑制INS-1细胞增殖。