四甲基偶氮唑盐法观察三水白虎汤含药血清对体外培养关节炎滑膜细胞增殖的影响

Effect of drug-contained serum of Sanshui Baihu Tang on the proliferation of in vitro cultured synoviocytes from arthritic synovitis: An observation by methyl thiazolyl tetrazolium assay

目的:采用四甲基偶氮唑盐比色法观察三水白虎汤含药血清对体外培养热痹证型关节炎滑膜细胞增殖的影响。方法:实验于2006-06/09在南方医科大学动物实验中心、中医药学院实验中心及组织工程研究中心完成。实验材料:滑膜组织取自南方医院脊柱外科收治的膝骨关节炎患者(女,68岁)行膝关节镜手术中切除的滑膜组织,患者知情同意。SPF级45d龄Wistar雌性大鼠15只,体质量(120±10)g。三水白虎汤中草药制剂(由南方医院中药房提供,主要成分水牛角、寒水石、白芥子、虎杖、鸡血藤等),来氟米特片(由美国欣凯公司提供)。实验分组:大鼠随机分为三水白虎汤组、来氟米特组以及生理盐水组,分别给予相应药液灌胃7d,制取含药血清。细胞培养实验共分4组,包括正常对照组以及三水白虎汤组、来氟米特组、生理盐水组3组含药血清组。实验干预:滑膜采自膝骨关节滑膜炎滑膜组织,采用体外细胞组织块培养技术,含药血清法接种培养。将第4代的滑膜细胞浓度为5×106L-1接种于板孔,在3组含药血清终浓度分别为10%,20%,30%,40%,50%下培养,正常对照组为完全培养液组。实验评估:①用四甲基偶氮唑盐检测法观察滑膜细胞的增殖。②锥虫蓝染色计数活细胞数,并进行B型细胞鉴定。结果:①滑膜细胞活性鉴定、计数及B型细胞鉴定:滑膜组织块培养后锥虫蓝拒染细胞计数为2×107,测得活性率达97%。免疫组织化学示Vimentin阳性,为B型滑膜细胞特征染色。②各组含药血清对滑膜细胞增殖的影响:生理盐水组和正常对照组的活细胞均明显增加;与生理盐水组和正常对照组比较,三水白虎汤组及来氟米特组的细胞增殖显著受抑致,活细胞数剧减(P<0.001)。在培养前5d,同一含药血清浓度下,三水白虎汤组随着培养天数增加,对细胞增殖的抑制作用增强,5d后抑制作用不再明显增加。同条件下比较活细胞数,三水白虎汤组和来氟米特组之间差异不明显(P>0.05),生理盐水组和正常对照组的活细胞数之间差异不明显(P>0.05)。结论:三水白虎汤含药血清对体外培养关节炎滑膜细胞增殖有显著抑制作用,抑制滑膜增生的途径可能为三水白虎汤治疗类风湿关节炎(热痹证型)的作用机制之一。

AIM： To observe the effect of drug-contained serum of Sanshui Baihu Tang （SBT） on the proliferation of synoviocytes of arthritis of heat arthralgia type cultured in vitro by methyl thiazolyl tetrazolium （MTT） assay. METHODS： The experiment was conducted in the animal experimental center, experimental center of Traditional Chinese Medicine College, and Institute Center of Tissue Engineering of Southern Medical University from June to September 2006. The synovial tissues were from one female patient of 68 years old, with ostarthritis of knee underwent arthroscope operation of knee joint in Department of Spinal Surgery of Nanfang Hospital. SBT preparation composed of comu bubali, calcite, white mustard seed, giant knotweed rhizome and caulis millettiae, was provided by the dispensary of traditional Chinese medicine of Nanfang Hospital, and leflunomide was provided by American Cinkate Corporation. Fifteen SPF grade female Wistar rats of 45 days old and （120±10） g were selected and randomly divided into SBT group, leflunomide group and normal saline group, which were intragastricaUy infused with corresponding drugs for 7 days to prepare the drug-contained serum. Cultured cells were divided into normal control group, and three drug-contained serum groups. The synoviocytes were cultured in vitro by drug-contained serums, and the fourth generation cells were inoculated into the plate at 5×10^6 cells/L with the drug-contained serums at the final concentrations of 10%, 20%, 30%, 40%, and 50%; meanwhile, the group cultured with the complete culture liquid served as the normal control group. The cell proliferation was analyzed by MTI assay; the viable cell count was analyzed by trypan blue staining, and type B cells were identified. RESULTS： （1）After culture, the trypan blue staining showed that cell count was 2×10^7, and the activity rate was 97%. Meanwhile, the immunohistochemistry suggested positive Vimentin, which was the feature of type B synoviocytes. （2）The viable cell count in the saline group and normal control group was significantly increased; on the contrast, the cell count of SBT group and leflunomide group was markedly reduced （P 〈 0.001 ）. Within the first five days of culture, the inhibition effect of SBT was increased along with the culture time at the same serum concentration, while the effect was not significantly increased afterward. There was no significant difference in the viable cell count between SBT group and leflunomide group under the same conditions （P 〉 0.05）, neither the saline group and normal control group （P 〉 0.05）. CONCLUSION： The proliferation of knee arthritic synoviocytes is suppressed significantly by drug-contained serum of SBT, which may be one of the action mechanisms of SBT to treat the rheumatoid arthritis of heat arthralgia type.