人工关节周围界膜白细胞介素6的表达及健骨二仙丸的干预效应

Interventional effect of Jiangu Erxian Wan on the expression of interleukin 6 in the interface membrane around joint prosthesis

目的:观察中药健骨二仙丸对体外培养人工关节假体周围界膜白细胞介素6表达的抑制作用,为健骨二仙丸防治人工关节假体周围骨溶解提供科学依据。方法:实验于2006-09/12在深圳市中医院中心实验室(国家级P2实验室)完成。①体外界膜组织培养:将备用的人工关节假体周围界膜(20g,取自右股骨颈骨折人工关节置换术后11年出现假体无菌性松动来深圳市中医院行翻修术患者,女性,74岁,对实验知情同意并经医院伦理委员会批准)放入Hank’s液中清洗后置于RPMI培养液中,然后将界膜标本用眼科剪剪成1mm3大小组织悬浮液。②含药血清制备:按每日中药健骨二仙丸生药12.0g/kg大鼠体质量灌胃(相当于临床剂量的6.25倍),每日固邦用量为1.04mg/kg体质量(相当于临床剂量的6.25倍)。2次/d,间隔5h,连续灌胃3d。末次灌药1h后,从腹主动脉取血,离心获取血清。③分组:取24孔培养板2块,分空白对照组、健骨二仙丸组、固邦组,各组又分别分100g/L,200g/L两个质量浓度亚组,共计6个组,每组8个培养孔。100g/L质量浓度组相应加入0.9mL组织悬液和0.1mL空白血清或健骨二仙丸、固邦含药血清。200g/L质量浓度组相应加入0.8mL组织悬液和0.2mL空白血清或健骨二仙丸、固邦含药血清。④实验评估:各组添加空白血清或含药血清后在体积分数为0.05CO2、37℃饱和湿度下培养72h,取上清液,用酶联免疫吸附法测定白细胞介素6的含量。上述界膜组织标本同时做细菌培养。结果:48个培养孔中的组织培养均成功,全部进入结果分析。①白细胞介素6水平:100g/L与200g/L空白对照组比较差异无显著意义[(97.113±11.989),(96.275±13.087)ng/L,P>0.05]。100g/L健骨二仙丸组[(92.288±10.397)ng/L]与空白对照组相比差异无显著性(P>0.05);200g/L健骨二仙丸组[(82.263±9.580)ng/L]低于空白对照各组(P<0.05)。100g/L与200g/L固邦组[(83.300±9.039),(79.338±11.118)ng/L]低于空白对照各组(P<0.05和P<0.01)。200g/L健骨二仙丸组与100g/L和200g/L固邦组比较,差异无显著性。②人工关节周围界膜组织细菌培养结果为阴性。结论:健骨二仙丸能够抑制磨损颗粒诱导的人工关节假体周围界膜细胞因子的分泌,进而阻止假体周围破骨细胞性骨溶解,对人工关节假体无菌性松动可能具有较好的防治作用。

AIM： TO observe the inhibition of Chinese herb Jiangu Erxian Wan （JGEXW） to the expression of interleukin 6 ilL-6） in the interface membrane of joint prosthesis cultured in vitro, so as to provide reference for the prevention of osteolysis with JGEXW. METHODS： The experiment was conducted in the Central Laboratory （P2 national laboratory） of Shenzhen Hospital of Traditional Chinese Medicine from September to December 2006. （1） The prepared interface membrane of joint prosthesis （20 g） was harvested from a female 74-year-old patient underwent overhauling in the hospital due to the aseptic loosening 11 years after prosthetic replacement of joint （agreed by the patient and permitted by the Hospital Ethics Committee）, and cultured in RPMI culture solution after washed by Hank＇s solution. Then the interface membrane was cut into 1 mm^3 pieces with the eye scissors to prepare the tissue suspension. （2）Rats were intragastricaUy infused with JGEXW at dose of 12.0 g crude drug/kg daily （6.25 times of clinical dose）, and alendronate sodium tablets of 1.04 mg/kg body mass （6.25 times of clinical dose）, twice daily, 5 hours interval for 3 days. One hour after the last administration, serum was harvested from blood of abdominal aorta by centrifugalization.（3）Two 24-well plates were divided into blank control group, JGEXW group, alendronate sodium tablets group, and each group was subdivided into 100 g/L and 200 g/L subgroups with 8 wells in each group. 100 g/L groups were added 0.9 mL tissue suspension and 0,1 mL serum or serum containing JGEXW and alendronate sodium tablets, respectively; 200 g/L groups were added 0.8 mL tissue suspension and 0.2 mL serum or serum containing JGEXW and alendronate sodium tablets, respectively. （4）After added various serums, every group was cultured at 37 ℃ with 0.05 volume fraction CO2 and saturated humidity for 72 hours. The content of IL-6 in supernatant fluid was detected by enzyme linked immunosorbent assay. Additionally, the above-mentioned interface membrane was bacterially cultured. RESULTS： All tissues of 48 wells were involved in the result analysis.（1）The IL-6 levels of 100 g/L and 200 g/L blank control groups were not statistically different [（97,113±11,989）, （96.275±13,087） ng/L, P 〉 0.05）, There was no significant difference in the IL-6 levels between 100 g/L JGEXW group and blank control groups [（92.288±10,397） ng/L, P 〉 0.05]; the levels of 200 g/L JGEXW group were lower than that of the blank control group [（82.263±9,580） ng/L, P 〈 0.05], The levels of 100 g/L and 200 g/L alendronate sodium tablets groups [（83.300±9.039）, （79.338±11.118） ng/L] were all lower than that of blank control group （P〈 0.05, P〈 0.01）, and no obvious difference was found between 200 g/L JGEXW group and 100 g/L and 200 g/L alendronate sodium tablets groups, （2）The bacterial culture of the interface membrane around joint prosthesis was negative. CONCLUSION： Chinese herb JGEXW could inhibit the secretion of cell factor of the interface membrane around joint prosthesis induced by wear debris, and prevent the osteolysis around the prosthesis, It has good preventive effect on the aseptic loosening of joint prosthesis.