【作者】 李显澎；

【导师】 樊粤光；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2009， 博士

【摘要】 1.研究目的儿童股骨头缺血性坏死,也称Legg-Calve-Perthes病,是骨科的疑难杂病之一,在1910年前后分别由Legg、Calve、Perthes报道后,国内外已进行各个方面的研究,一直在不断加深对其认识中。儿童股骨头坏死的病因存在多种学说,在研究儿童股骨头坏死治疗方法过程中,针对其可能病因的治疗方法有了很多的报道,但是针对不能肯定的病因而设计的手术,有无必要,有无作用和副作用等问题有待深思,有待进行大量病例的长期随访和科学的疗效评价,现多数学者主张病因未明确前不宜选用创伤大过程复杂的手术。祖国医学认为该病病机为肝肾不足,髓海空虚,不能滋养骨骼,如存在感受外邪等诱因,致使瘀血凝滞,经脉受阻,气血不通,这也是股骨头坏死的发病机理。股骨头坏死关键在于缺血。肾阴亏损、气滞血瘀是儿童股骨头坏死的常见病机,治疗上应以补益肝肾,活血化瘀,行气止痛为法。中药生脉成骨胶囊是治疗股骨头坏死的药物,在实验和临床上已经做了大量的研究,可以改善微循环、抑制细胞成脂、促进血管生成,治疗股骨头坏死具有较好的疗效。根据病理改变的规律设计的治疗方法,已经受到国内外众多学者的重视,在自然修复的整个病理过程中,积极地采取避免股骨头变形的措施,比如髋外展支架,截骨包容法,可以根据生物性塑形使得髋关节达到匹配,具有较好的疗效和应用前景。骨髓间质干细胞因其本身具有强大增殖能力和多向分化潜能,在修复创伤领域研究具有重要的意义。本论文拟研究分离培养骨髓间质干细胞,然后采用补肾活血化瘀中药生脉成骨胶囊进行培养,观察对骨髓干细胞是否有增殖作用和促进成骨,初步分析这种作用可能是该药的作用机理之一。再回顾性研究对比多种方法处理儿童股骨头坏死的结果,即未系统治疗、髋外展支架治疗、中药配合支架治疗并进行初步分析,试探讨用中药和包容疗法治疗儿童股骨头坏死的应用价值。2.研究方法研究一:骨髓间质干细胞的分离培养和成骨分化的鉴定。1.分离大鼠骨髓间质干细胞,在37℃、5g/LCO₂、饱和湿度条件下体外培养。培养后的第3天更换培养液以更新细胞代谢环境。以后每3 d换液1次,去除未贴壁的细胞,待细胞汇合约80%时,用2.5g/L胰蛋白酶（0.4g/L EDTA）消化,按1:2传代培养。2.倒置显微镜下观察细胞生长的形态特性、取生长良好的2、3代细胞,2.5g/L胰蛋白酶消化,1×10⁴/mL接种于24孔板,每天各取3孔消化计数,每孔计数3次,连续7d。以培养时间为横坐标,细胞数为纵坐标,描绘生长曲线。3.用CD34,CD44,CD54抗体免疫法,钙钴法,矿化结节染色检测骨髓间质干细胞成骨作用。研究二:生脉成骨胶囊对骨髓间充质干细胞增殖作用和成骨分化的实验研究。1.制备生脉成骨胶囊含药血清:给予大鼠生脉成骨胶囊1 g/kg灌胃,于首次灌胃后2 h再次灌胃,末次灌胃1 h后腹主动脉采血,离心3000 r/min,20min。收集上清,过滤除菌。用L-DMEM配成含100 g/L含药血清的培养基。另一组以生理盐水灌胃、采血、培养基配制,配制成含空白血清的培养基。2.选择合适密度的骨髓间质干细胞,给予用血清浓度10%的生脉成骨胶囊血清和空白组（即生理盐水灌胃）对骨髓间质干细胞在24孔板中进行培养,每天各取3孔消化计数,每孔计数3次,连续7d。以培养时间为横坐标,细胞数为纵坐标,描绘生长曲线。3.于第7天时吸取出培养液,用PBS冲洗3次,每次1min,中性福尔马林室温固定10min蒸馏水冲洗3次,加入新鲜的孵育液37℃孵育6h,蒸馏水冲洗3次,20g/L硝酸钴水溶液浸泡5min,蒸馏水冲洗3次,10g/L硫化铵水溶液5min,水洗,中性树胶固定,标记,镜下观察。12d时的吸取出培养液,PBS清洗3次,950g/L乙醇固定30min,PBS清洗3次,1g/L茜素红染色,双蒸水冲洗3遍,中性树胶固定,标记,镜下观察。研究三:儿童股骨头坏死的临床研究1.采用临床回顾性研究为主的方法收集广州中医药大学第一附属医院髋中心2004年8月～2007年1月诊断为儿童股骨头坏死的患儿,进行病例随访,对治疗方法进行分类,有如下三类:未系统治疗、髋外展支架治疗、补肾活血化瘀中药生脉成骨胶囊配合髋外展支架治疗。用统计学的方法分析各组病例的年龄,Catterall分型,骨骺突出指数,VAS疼痛评分,Stulberg分型,Mose环标准,临床症状,以及各组治疗的有效率、优良率情况,然后将有效率行×列表的X²检验观察相关治疗方法的结果评定。3.研究结果研究一:骨髓间质干细胞的分离培养和成骨分化的鉴定。1.全骨髓培养法可以获得较好的骨髓间质干细胞,体外培养具有良好的增殖能力,细胞能在4～7d可完成原代培养。P2代细胞生长迅速,以1:3传代,3天即可铺满瓶底。形成均一性良好的单层细胞,在诱导液诱导下可见成骨分化。2.实验发现MSCs的生长性状基本稳定,二代细胞的形态、生长曲线无显著性差异,细胞为均一的成纤维细胞样,潜伏适应期在第1～2天（约24h）,对数生长期为第3～5天（约72h）,以后进入平台期,在对数生长期,群体倍增时间34h,传代周期约6d,每传代1次细胞增加约2倍。3.免疫法结果显示在MSCs膜上呈深红色,不透光为MSCs特性CD44（+）CD54（+）,而不表达造血干细胞的特性CD34（-）。经诱导后的细胞,碱性磷酸酶染色明显,胞质中阳性反应呈现灰黑色颗粒或块状沉淀,ALP活性部位呈棕黑色。茜素红染色可见红色的矿化结节颗粒,颗粒大小不均一,提示有矿化基质沉积。研究二:生脉成骨胶囊对骨髓间充质干细胞增殖作用和成骨分化的实验研究。1.采用生脉成骨胶囊血清培养骨髓间质干细胞,细胞计数法测定和对照组对比可见细胞增殖明显。2.经中药血清培养后的细胞,碱性磷酸酶染色明显,胞质中阳性反应呈现灰黑色颗粒或块状沉淀,茜素红染色可见红色的矿化结节颗粒,颗粒大小不均一。研究三:儿童股骨头坏死的临床研究。在临床研究中,共有75例78髋纳入,其中未系统治疗有18髋18例,髋外展支架治疗有18例19髋,中药配合支架治疗有39例41髋,经过3年随访,结果表明组内髋部疼痛VAS评分有差异（P＜0.05）。股骨头的外形和骨骺的高度经过中药配合髋外展支架治疗恢复得较好。按Mose环标准,好8髋,可22髋,差11髋。头臼的匹配情况按照Stulberg分型,三组之间存在差异。整体的疗效评价用临床症状评价,三组之间以髋的活动度和步态经过比较存在差别,采用生脉成骨胶囊配合髋外展支架治疗儿童股骨头坏死较好,优良率为68.3%,有效率82.9%（P＜0.05）。4.结论4.1用全骨髓培养法可以获得较好的骨髓间质干细胞,具有增殖能力和在诱导下具有定向分化能力。4.2通过补肾活血化瘀中药生脉成骨胶囊含药血清的制备,在体外培养的情况下,和对照组相比,骨髓间质干细胞增殖明显,并且有成骨作用,这可能是该中药在临床上治疗股骨头坏死的机理之一。4.3应用中药生脉成骨胶囊配合髋外展支架治疗儿童股骨头坏死病这一治疗方法有较好的疗效。更多还原

【Abstract】 1.Objective:Ischemic necrosis of children femoral head,also called Legg-Calve-Perthes disease was describled by Legg、Calve、Perthes three professors in 1910.Many experts have studied much about every aspect of this disease since then,they have make much progress.There are various kinds of etiologies about this disease,many treatments based on etiologies had been reported during the study process,however the operation based on the possible etiologies had brought a lot of controversy such as the following questions: is it necessary ? is it effective? Do it have side effect?.these debate need to be considered seriously,to be supported by long-term follow-up of a lot of cases and scientific effect evaluation,nowadays most scholars oppose that complex and traumatic operations would not be used untill the etiology is clear.Chinese medicine consider this disease is caused by deficiency of the kidney and liver,bone marrow is so few that it can not nourish the bone,if the external pathogen invade,it will cause the condition of the stagnation of qi and stasis of blood,meridian obstruction.The pathogenesis of necrosis of femoral head is lack of blood.Deficiency of kidney and liver is the common pathogenesis,we should invigorate the kidney and liver,activate blood circulation to dissipate blood stasis,move the qi to stop the pain.Sheng Mai Cheng Gu Capsule is a Chinese medicine for necrosis of the femoral head,there are many experiments and clinical application reports that have indicated its effectiveness.More and more scholars have pay more attention to the treatments based on the discipline of pathological change.The methods that prevent the femoral head deformity such as hip abduction orthosis and osteotomy containment treatment should be undertaken when it is waiting for the natural recovery to reach the matching state of hip and acetabular according to the biological shape,the method have the good effect and application future.Bone marrow mesenchymal stem cells study is important and meaningful in recovery and traumatic study field because it can proliferate again and again,can differentiate into the osteolbast.This study is planning to isolate and culture the bone marrow mesenchymal stem cells,then use the Chinese medicine Sheng Mai Cheng Gu Capsule that can invigorate the kidney and liver,activate blood circulation to dissipate blood stasis to culture the cells to observe whether the medicine can promote the cell proliferation and osteoblast or not,and We shall analyze the result,ln the clinical study,we compared and analyzed the outcomes of different treatments such as no systematic treatment、hip abduction orthosis,chinese medicine with orthosis to explore the medicine mechanism and clinical application of chinese medicine combined with hip abduction orthosis.2.MethodsStudy one:isolation and culture of MSCs and its osteoblast identification.1.bone marrow mesenchymal stem cells were isolated from Rat lower extremity, cultured in the condition of 37℃、5g/LCO₂、saturated humidity.The medium was changed to renew the metabolic environment and discard the non-adherent cells every three days.When the cell convergence were about 80%,it was digested with 2.5g/L trypsin（0.4g/L EDTA） at the ratio of 1:2.2.Cells shape was observed through inverted microscope.P2 and P3 cells were digested with 2.5g/L trypsion,vaccinated in twenty four hole culture plate with 1×10⁴/mL density,the number of cells were calculated every day for seven days and the growth curve was made.3.The CD34,CD44,CD54 antibody immunosorbent assay,calcium-cobalt method,mineralized nodules method were used to evaluate the osteoblast effect of MSCs.Study two:Experimental study of effect of Sheng Mai Cheng Gu Capsule serum on MSCs proliferation and osteoblast differentiation.1.Production of Sheng Mai Cheng Gu Capsule serum:The rats were feed with 1 g / kg Sheng Mai Cheng Gu Capsule,feed them in two hours.The blood was collected through Abdominal aorta in one hour,Centrifugal 3000 r / min,20min, the upper layer of liquid was collected,Filtered for sterilization,the 100 g / L medicine serum medium with L-DMEM were produced,the other group used the saline.2.The appropriate density bone marrow mesenchymal stem cells was choosed, then the Sheng Mai Cheng Gu Capsule serum medium and saline medium were used to culture cells,the number of cells were measureed each day by counting to observe the proliferation of stem cells.3.Medium was removed after seven days,washed three times with PBS,one minute each time,neutral formalin for fixation for ten minutes,washed with distilled water for three times,added fresh incubation medium for six hours,washed with distilled water,,then 20g/L Cobalt nitrate water was used for immersion for five minutes,washed with distilled water,then used lOg/L Ammonium sulfide water for staining for five minutes,washed with distilled water,neutral gum was used for fixation,and observed through microscope. the medium was removed after twelve days,washed three times with PBS,950g/h alcohol was used for immersion for thirty minutes,washed three times with PBS,then the 1g/L Alizarin red staining was used,neutral gum was used for fixation and observed through microscope.Study three:Clinical study of Perthes disease.1.A retrospective study of children femoral head necrosis from August, 2004 to Jaunary,2007,in the First Affiliated Hospital of GuangZhou Unive rstiy of Chinese Medicine was made.There were three groups:no systematic treatment,hip abduction orthosis,Chinese medicine with orthosis,we colle cted and sorted out imformation,analyzed the effectiveness rate, excellent and good rate according to the Stulberg,Mose circle criteria, epiphyseal extrusion,clinical symptoms,then analyzed with the Chi-squar e test.3.Results: Study one:isolation and culture of MSCs and its osteoblast identification.1.Bone marrow mesenchymal stem cell could be got through the all bone marrow culture,the cells have excellent ability to proliferation in vitro,the cell can finish the original culture in four-seven days.F2 cells growed rapidly, passed at the ratio of 1:3,the cells can cover the bottom of bottle in three days and form the good mono-layer cells.The cells can be induced into osteoblast by induction medium.2.The study indicate the growth state of MSCs is stable,there is no difference between P2 and P3 in shape and growth curve.The cells are even and fibroblast-like.Potential adaptation period are 1～2 days,logarithmic phase are the third or the fifth day,then come to plateau.In the logarithmic phase,colony multipliy for thirty four hours,passage period is about six days, the number of cells will double through each passage.3.immunosorbent assay indicated membrane of MSCs is dark red,opaque in CD44（+）CD54（+）,but don’t showed the feature of hematopoietic stem cell.After induction,ALP staining is obvious that grey and black particles or massive Precipitation within the Cytoplasmic.Alizarin red staining can show red Nodular particles indicated mineralized matrix deposition.Study two:Experimental study of effect of Sheng Mai Cheng Gu Capsule serum on MSCs proliferation and osteoblast differentiation.1.Sheng Mai Cheng Gu Capsule serum can get more cells through proliferation than the saline group.2.After the Chinese medicine serum medium culture,ALP staining was cle ar,there were grey and black particles or massive Precipitation within th e Cytoplasmic.Alizarin red staining can show red Nodular particles.Study three:Clinical study of Perthes disease.1.In the clinical study,we had seventy five cases（seventy eight hips） at last.No systematic treatment group had eighteen cases（eighteen hips）,hip abduction orthosis group had eighteen cases（nineteen hips）,Chinese medicine combining orthosis group had thirty nine cases（fourty one hips）.After three years follow-up,there was difference in VAS hip pain and outcome（P＜0.05）,the Chinese medicine combining orthosis group hava a better outcome.The clinical results showed there was significant difference in three groups,the Sheng Mai Cheng Gu Capsule combined with abduction orthosis gourp had the best results.4.Conclusions:4.1 We can have the BMSCs by using the all bone marrow culture method.The cells can be induced into osteoblast by induction medium. 4.2 Sheng Mai Cheng Gu Capsule serum can promote the stem cells proliferation and induce them into osteoblasts in vitro compared to saline group,this may be one mechanism of this medicine that can treat the necrosis of children femoral head.4.3 Applying the the Sheng Mai Cheng Gu Capsule combined with hip abduction orthosis to the treatment of Perthes disease is a good and effective method,更多还原