大鼠骨骼肌脱细胞处理方法的优化研究

OPTIMAL METHOD FOR RAT SKELETAL MUSCLE DECELLULARIZATION

目的探讨SD大鼠骨骼肌脱细胞的优化处理方法。方法SD大鼠16只,雌雄不限,体重180～200g。取其中10只大鼠36条骨骼肌肌束,随机分为3组,正常组(A组,n=4):不作脱细胞处理;时间组(T组)及浓度组(C组)按照低渗-去垢剂方法进行脱细胞处理(n=16),其中T组十二烷基磺酸钠(sodium dodecyl sulfate,SDS)浓度均为1.0%,根据处理时间不同(24、48、72、96h),分为T1、T2、T3、T4组(n=4);C组处理时间均为48h,根据SDS浓度不同(0.5%、1.0%、1.5%、2.0%)分为C1、C2、C3、C4组(n=4)。取各组材料行HE染色观察及羟脯氨酸含量检测,并根据结果筛选最优脱细胞处理条件。另取6只SD大鼠14条完整肌束,随机取7条按照最优条件进行脱细胞处理(实验组),余7条作为正常对照(对照组),对肌束行大体观察、Masson染色及生物力学检测。结果HE染色显示T1、T2、C1、C2、C3组肌纤维与A组无差异;C4组肌纤维部分脱除;T3组肌纤维完全脱除且基膜结构保留完整;T4组肌纤维完全脱除但基膜结构部分被破坏。羟脯氨酸含量检测:A组与C1、C2、C3、T1及T2组比较,差异无统计学意义(P>0.05);A组与C4、T3和T4组比较,差异有统计学意义(P<0.05);C4组与T3、T4组比较,差异有统计学意义(P<0.05);T3组和T4组比较,差异无统计学意义(P>0.05)。根据HE染色及羟脯氨酸含量检测实验结果筛选出最优脱细胞处理条件为4℃、1.0%SDS、72h。大体观察:实验组与对照组比较肌束颜色变浅,呈半透明状,且结构相对较松散。Masson染色观察:实验组胶原纤维连续性良好,较对照组结构疏松。生物力学测试:实验组及对照组最大破坏载荷分别为(1.38±0.35)N及(1.98±0.77)N,最大拉伸位移分别为(3.19±3.23)mm及(3.56±2.17)mm,两组以上指标差异均无统计学意义(P>0.05)。结论应用1.0%SDS,于4℃条件下对大鼠骨骼肌经震荡处理72h可得到完全去除肌纤维且ECM保留完好的脱细胞基质。

Objective To investigate an optimal method for SD rat skeletal muscle decellularization. Methods Sixteen SD rats （male and female） weighing 180-200 g were used. Thirty-six skeletal muscle bundles obtained from 10 rats were randomly divided into 3 groups： normal group （group A, n=4） received non-decellularization; time group （group T, n=16） and concentration group （group C, n= 16） underwent decellularization using hypotonic-detergent method. Concentration of sodium dodecyl sulfate （SDS） was 1.0% for T group, which was subdivided into groups T1, T2, T3 and T4 （n=4 per subgroup） according to different processing durations （24, 48, 72 and 96 hours）. Group C was treated for 48 hours and subdivided into groups C1, C2, C3 and C4 （n=4 per subgroup） according to different SDS concentrations （0.5%, 1.0%, 1.5% and 2.0%）. The muscle bundles of each group underwent HE staining observation and hydroxyproline content detection in order to get the optimal decellularization condition. Seven of 14 complete skeletal muscle bundles obtained from 6 SD rats were treated with the optimal decellularization condition （experimental group）, and the rest 7 muscle bundles served as normal control （control group）. The muscle bundles of each group were evaluated with gross observation, Masson staining and biomechanical test. Results HE staining： there was no significant difference between groups T1, T2, C1, C2 and C3 and group A in terms of muscle fiber; portion of muscle fibers in group C4 were removed; muscle fibers in group T3 were fully removed with a complete basement membrane structure; muscle fibers of group T4 were fully removed, and the structure of basement membrane was partly damaged. Hydroxyproline content detection： there was no significant difference between group A and groups C1, C2, C3, T1 and T2 （P 〉 0.05）; significant difference was evident between group A and groups C4, T3 and T4 （P 〈 0.05）; the difference between group C4 and groups T3 and T4 was significant （P 〈 0.05）; no significant difference was evident between group T3 and group T4 （P 〉 0.05）. The optimal decellularization condition was 4 ℃, 1.0% SDS and 72 hours according to the results of HE staining and hydroxyproline content detection. Gross observation： the muscle bundles of the experimental group were pallid, half-transparent and fluffier comparing with the control group. Masson staining observation： the collagen fibers of the experimental group had a good continuity, and were fluffier comparing with control group. Biomechanics test： the maximum breaking load of the experimental group and the control group was （1.38 ± 0.35） N and （1.98 ± 0.77） N, respectively; the maximum extension displacement of the experimental group and the control group was （3.19 ± 3.23） mm and （3.56 ± 2.17） mm, respectively; there were no significant differences between two groups （P 〉 0.05）. Conclusion Acellular matrix with intact ECM and complete removal of muscle fibers can be obtained by oscillatory treatment of rat skeletal muscle at 4℃ with 1% SDS for 72 hours.