【作者】 欧阳建江；

【导师】 刘庆思；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2012， 博士

【摘要】 目的：1通过随机设计的临床研究,观察温和灸对肾阳虚型绝经后骨质疏松症骨转换指标、临床骨痛及肾阳虚证候的影响,探讨其防治骨质疏松症临床效果及作用机制。2通过实验研究来检测艾灸血清对骨质疏松症成骨细胞增殖率及凋亡情况,从细胞分子水平探讨其治疗骨质疏松症的作用机理。方法：1临床实验：运用温和灸疗法治疗肾阳虚型绝经后骨质疏松症,共入选研究病例90例,随机分为三组,每组各30例,研究结束后剔除不符合研究要求及脱落病例,共纳入统计病例87例,分别为：钙剂组28例,中成药组30例,温和灸组29例。钙剂组患者予以口服钙尔奇碳酸钙D3片,1片/次,1次/日。中成药组患者在钙剂组的治疗基础上加服仙灵骨葆胶囊,每次3粒,每日两次,温和灸组在钙剂组的治疗基础上循经取穴行温和灸疗法,隔日一次,取穴为3个主穴+3个配穴,主穴如下：脾俞、胃俞、肾俞。配穴选用命门、腰阳关、至阳。三组均连续治疗三个月,观察治疗前后肾阳虚证侯的变化及临床骨痛改善情况、骨密度、体表红外热图皮温的改变,并按中医临床疗效评定标准评价其疗效,空腹抽血检测治疗前后患者的骨形成相关指标：血清骨钙素(BGP)、血清骨碱性磷酸酶(BALP)。骨吸收相关指标抗酒石酸酸性磷酸酶5b(TRACP-5b)、骨保护素(OPG)。骨矿化指标血钙(Ca)、血磷(P)。治疗前后各指标比较组内采用配对样本t检验,组间差异用方差分析,行LSD、SNK法进行统计分析。2体外培养细胞实验：通过观察大鼠艾灸血清对成骨细胞增殖与凋亡的影响,来探讨温和灸治疗骨质疏松症的细胞生物学机理。取10只出生24小时的新生SD大鼠无菌取颅盖顶骨,用单纯胶原酶消化法行成骨细胞培养,制成DMEM培养液重悬细胞,吹打均匀后接种到多个37℃、5%CO2饱和湿度的培养瓶中进行培养,进行成骨细胞HE染色观察及ALP染色鉴定,取二代细胞进行实验。将6月龄SD雌性大鼠随机分成2组：艾灸血清组、空白血清组,每组10只,艾灸血清组经用陈艾绒制成的艾条在大鼠背俞穴(脾俞、胃俞、肾俞、命门、腰阳关、至阳)行温和灸法,每天一次,每穴10分钟,连续21天,空白血清组大鼠除不作艾灸治疗外,余处理同艾灸血清组。制备艾灸血清和空白血清,同组血清混合后过滤除菌,56℃、30分钟水浴灭活,无菌冻存管分装、标记后-30℃冻存备用。取艾灸血清加入DMEM培养基中,制成含10%艾灸血清的培养液,同样取空白血清加入DMEM培养基中,制成含10%空白血清的培养液。将培养液分为空白对照组(含10%FBS、DMEM)、血清对照组(含10%空白血清、DMEM)和艾灸血清组(含10%艾灸血清、DMEM)三组进行培养,分别在24小时、48小时培养结束后,用MTT法检测成骨细胞增殖活性。按不同培养液条件分为空白对照组(含DMEM、10%FBS)、凋亡诱导组(DMEM.10%FBS、40ng/mlTNF-α),血清对照组(DMEM、10%空白血清、40ng/mlTNF-α)和艾灸血清组(DMEM、10%艾灸血清、40ng/mlTNF-α)四组,继续二代细胞培养,培养在48小时后完成,做HE、DAPI荧光染色,通过荧光倒置显微镜下检测成骨细胞凋亡情况。结果：1临床研究表明肾阳虚证候积分测定结果：治疗前后比较,钙剂组肾阳虚证候总积分比较无明显差异(P=0.676>0.05),中成药组畏寒肢冷证候积分无明显差异(P=0.083>0.05),余指标均有明显差异(P<0.01),其中以夜尿频多及疲乏无力证候改变为明显(P=0.001及P=0.003),温和灸组以畏寒肢冷、大便溏薄积分降低为明显(P=0.003及P=0.001),余证候积分P<0.05。治疗后组间方差分析两两比较,中成药组、温和灸与钙剂组在腰背疼痛及肾阳虚总积分改善方面有明显差异(P<0.05),温和灸与中成药组比较仅畏寒肢冷证候指标有显著差异(P=0.002<0.01),余证候指标相比较无明显差异(P>0.05)。临床VAS测定结果：治疗前后比较,钙剂组对于患者主观腰背疼痛症状无好转,VAS积分无明显改变,而中成药组及温和灸组均有明显好转(P=0.000<0.01)。治疗后三组组间比较,钙剂组VAS积分变化较小,中成药组与温和灸组相比,VAS积分下降幅度较大(P=0.015<0.05)。红外热像皮温值测定结果：结果显示,各组治疗前比较局部温度无明显差异,组内治疗前后比较,温和灸组局部温度明显升高(P=0.001<0.01),其他两组无明显差异。组间比较,温和灸组患者较其他两组患者局部温度增高,具有显著差异性(与钙剂组比较P=001,与中成药组比较P=0.014),钙剂组与中成药组无明显差异(P=0.389>0.05)。中医临床疗效测定结果：温和灸组临床显效占31.0%,总有效率86.2%。中成药组显效占33%,总有效率86.7%,温和灸组和中成药组疗效无明显差异,但均明显优于钙剂组(P=0.007<0.01)。骨密度测定结果：骨密度各组间治疗后比较,均无显著差异。骨转换指标的测定结果：三组治疗前比较,各指标无明显差异,具有可比性(P>0.05),BGP组内比较,中药组及温和灸组有显著差异(P=0.000<0.01),钙剂组无明显差异。BGP组间比较,温和灸组较钙剂组显著增加(P=0.004<0.01),较中成药组明显无明显改变(P=0.075>0.05)。BALP组内比较,钙剂组治疗前后无明显差异,中成药组BALP有所增加(P=0.001<0.01),温和灸增高的幅度略大(P=0.000<0.01)。组间与钙剂组相比较,均存在一定的差异性(P<0.01>0.05),中成药组和温和灸组增高值相比较无明显差异,都在正常范围(P=0.306)。三组TRACP5b均有下降,钙剂组治疗前后比较无差异,中成药组及温和灸治疗前后相比较,具有显著差异(P<0.01),三组治疗后比较,钙剂组与中成药组比较存在显著差异性(P=0.005<0.01),与温和灸组比较有差异(P=0.013<0.05),中成药组与温和灸组比较无明显差异(P=0.775>0.05)。OPG组内比较,中成药组存在显著差异(P=0.001<0.01),温和灸存在显著差异(P=0.002<0.01)。但治疗后组间比较,温和灸组与钙剂组相比,有一定程度的下降(P=0.044<0.05),中成药组与钙剂组比较有差异(P=0.046<0.05)。三组治疗前后及各组间比较,血清钙、磷水平无明显差异(P>0.05)。2实验研究表明,空白对照组成骨细胞凋亡率及凋亡细胞均值较其他三组少(P=0.000),艾灸血清组成骨细胞凋亡率、凋亡细胞均值与凋亡诱导组及血清对照组比较,有明显差异(P<0.05),血清对照组与凋亡诱导组比较无明显差异(P>0.05)。三组在24小时时段成骨细胞增殖OD570值无明显差异(P>0.05),在48小时后各组OD570均有明显增加,艾灸血清组增殖幅度较大,与空白对照组及血清对照组比较,有显著差异(P<0.05),血清对照组与空白对照组比较无明显差异(P>0.05)。在不同时段比较,空白对照组和血清对照组OD570值均增加,但组内比较尚无明显差异(P>0.05),艾灸血清组不同时段组内比较,成骨细胞OD570值有明显增加(P<0.01)。结论：1温和灸能明显改善肾阳虚绝经后骨质疏松症患者临床症状,缓解骨质疏松症骨痛,并优于其他治疗组。2温和灸可明显提高肾阳虚型骨质疏松症腰部疼痛部位及周围的红外热像皮温值,有效改善肾阳虚证候。3温和灸可提高骨形成相关指标水平,降低骨吸收相关指标,降低肾阳虚型PMOP的骨转换。4艾灸血清能促进成骨细胞增殖,可减少TNF-α诱导的成骨细胞凋亡数量。更多还原

【Abstract】 Objective1Through a randomized clinical study designs, to observe the effect of Bone turnover markers, Clinical pain and Kidney yang deficiency syndrome on postmenopausal osteoporosis. to analysis the prevention of osteoporosis clinical efficacy and mechanism of action.2Through the experimental research to detect the effect of moxibustion serum on osteoporosis osteoblast proliferation and apoptosis, From the cellular and molecular level to investigate its role in the treatment of osteoporosis mechanism.Methods1Clinieal study:A total of90patients with postmenopausal osteoporosis in kidney yang deficiency were assigned, and randomly divided into3groups,30cases in each group, the end of the study does not meet the research requirements and elimination after shedding case, were included in the statistical case87examples, respectively:28cases in the Calcium agent group,30cases in the Proprietary Chinese medicine group and29cases in the mild moxibustion group. Patiets in the Calcium agent group were treated by1Calcium carbonate tablets D3once daily, Patiets in the the Proprietary Chinese medicine group were treated by3Xianlinggubao capsules twice daily and1Calcium carbonate tablets D3once daily. Patiets in the mild moxibustion group were treated by mild moxibustion on the The main points as Pishu point, WeiShu point, ShenShu point and acupoints as Mingmen acupoint acupoints, YaoYangGuan acupoint, ZhiYang acupoint Once every other day and1Calcium carbonate tablets D once daily. All the patients were treated for three months. To Observe the change of Kidney yang deficiency syndrome, Clinical pain, Bone mineral density and Body surface skin temperature of infrared thermograph. According to TCM clinic curative effect evaluation of its curative effect. Meanwhile to determinate patients with bone formation index such as Osteocalcin(BGP), serum bone alkaline phosphatase(BALP), bone resorption index such as tartrate-resistant acid phosphatase5b (TRACP-5b), osteoprotegerin (OPG),bone mineralization indicators serum calcium(Ca), phosphorus(P). Comparison of indexes before and after treatment in group was used with paired sample t test, the differences between groups was used with analysis of variance, LSD, SNK method for statistical analysis, with P<0.05was considered statistically significant criteria.2In vitro experiment:Through the observation of moxibustion serum on rat osteoblast proliferation and apoptosis, on mild-warm moxibustion for treatment of osteoporosis in cell biological mechanism.10born only24hours of neonatal SD rat calvarial bone aseptic were taken. DMEM medium heavy suspension cells came from Using pure collagenase digestion method line of osteoblasts culture, after All culture osteoblasts were inoculated into a plurality of37℃,5%CO2saturated humidity flask uniformed by Wind and percussion. HE staining and ALP staining identification were Conducted, experiments were using of the two generation cell. June old female SD rats were randomly divided into2groups, Each group of10. The rats of moxibustion serum group were treated with gentle moxibustion by moxa cone made by old argy wormwood leaf in rat Back-shu points once a day, each of10minutes for21days, To preparated moxibustion serum and blank serum. The same group of serum after mixing, filtration,56degrees,30minutes bath inactivation aseptic packaging, freezing tube, mark-30℃of cryopreserved standby. Using moxibustion serum added in DMEM medium containing10%, made of moxibustion serum medium, also take blank control group rats serum made with10%serum controls medium. The culture fluid is divided into the blank control group (10%FBS, DMEM), Serum control group (including10%blank serum, DMEM) and moxibustion serum group (including10%moxibustion serum, DMEM) in three groups were cultured respectively, in24hours,48hours after the cultivation,Use MTT method to detect the activity of osteoblast proliferation. According to different culture conditions were divided into control group(DMEM,10%FBS), Apoptosis induced by group (DMEM,10%FBS,40ng/ml TNF alpha), The control group, serum (DMEM,10%blank serum,40ng/ml TNF alpha)and moxibustion serum group (DMEM,10%,40ng/ml TNF moxibustion serum alpha) in four groups, continue to the two generation in cell culture, culture at48hours after completion, do DAPI fluorescence staining, Through an inverted fluorescence microscope osteoblast apoptosis.Result1In clinical studies have shown as follows:Kidney yang deficiency syndrome integral determination results:Calcium group of kidney-yang deficiency syndrome total no difference (P=0.676>0.05), Chinese medicine group of aversion to cold syndrome scores showed no significant difference(P=0.083>0.05), More than indicators have obvious differences (P<0.01), with nocturia and fatigue without evidence of climate change (P=0.001and P=0.003), Mild moxibustion group with aversion to cold, thin integral stool pond is reduced to significantly(P=0.003andP=0.001), P<0.05points more than syndrome. After treatment between group variance analysis comparison of two two, Chinese medicine group, mild moxibustion group and calcium in low back pain and kidney yang deficiency total integral improvement have obvious difference(P<0.05), Mild moxibustion and medicine group comparison only aversion to cold syndrome indices are significantly different(P=0.002<0.01), Other syndromes indices compared with no significant difference (P>0.05). Clinical result of measurement of VAS:Comparison before and after treatment, calcium group for low back pain patients with subjective symptoms do not improve, VAS integral has no obvious change, and Chinese patent medicine group and mild moxibustion group were improved significantly(P=0<0.01). Between group after treatment between the three groups, calcium group VAS integral small changes, Chinese medicine group and mild moxibustion group compared with the VAS integral, a larger decrease(P=0.015<0.05).Infrared thermography of skin temperature measurement results:The results showed, each group before treatment comparison of local temperature had no significant differences within the group, after treatment, mild moxibustion group of local temperature increased significantly (P=0.001<0.01), the other two groups had no significant difference. Comparison between groups, mild moxibustion group of patients than in the other two groups of patients with localized temperature increases, with significant difference (calcium group P=001, P=0.014compared with traditional Chinese Medicine), calcium group and the Chinese medicine group had no significant difference(P=0.389>0.05). Clinical effect of Chinese medicine determination results:Clinical effect of mild moxibustion group accounted for31%, the total efficiency of86.2%. Chinese patent medicine group was33%, the total efficiency of86.7%, Mild moxibustion group and the traditional Chinese medicine curative effect had no significant difference, But were significantly better than the calcium group (P=0.007<0.01).Determination of bone mineral density BMD results:the group after treatment, There were no significant differences.Bone turnover markers determination results:The three groups before treatment comparison, each index showed no significant differences, with comparable (P>0.05), BGP group, Chinese medicine group and mild moxibustion group were significantly different(P=0<0.01), calcium group had no significant difference. BGP comparison between groups, mild moxibustion group than the calcium group increased significantly(P=0.004<0.01),a proprietary Chinese medicine group was significantly (P=0.075>0.05). BALP group, calcium groups before and after treatment was not significantly different,proprietary Chinese medicine group BALP increased (P=0.001<0.01), Mild moxibustion increased amplitude slightly larger (P=0<0.01).Between group and calcium compared, there are some differences(P<0.01). Chinese medicine and moxibustion group significantly increased compared to the value have no significant difference,both in the normal range(P=0.306).Three groups of TRACP5b decreased calcium groups before and after treatment, no differences, Chinese medicine and moxibustion group before and after treatment are compared, have significant difference (P<0.01), the three group after treatment, calcium group and there are significant differences between Chinese medicine group(P=0.005<0.01), and mild moxibustion group comparison difference(P=0.013<0.05), Chinese medicine group and mild moxibustion groups have not obvious difference. OPG group compared esultst:there were significant differences of proprietary Chinese medicine group, mild moxibustion has significant difference. But after treatment were compared between groups, mild moxibustion group and calcium group, have a certain degree of down(P=0.044<0.05), Chinese medicine group and calcium group differences(P=0.046<0.05).The three groups before and after treatment and compared between groups, serum calcium, phosphorus levels have no significant difference.2Experimental studies have shown, the blank control component of bone cell apoptosis rate and cell apoptosis of mean lower than the other three groups, moxibustion serum composition of bone cell apoptosis, apoptosis and apoptosis induced by serum mean group and the comparison group, there are significant differences (P<0.05), serum controls and apoptosis induced by group comparison no significant difference(P>0.05). Three groups in the24hours of osteoblast proliferation od no difference (P>0.05), At48hours after each OD570obviously increased, The proliferation of moxibustion serum group greatly, and the blank control group, and serum in comparison to the control group, there were significant differences (P<0.05), Serum control group compared with the control group no significant difference(P>0.05). In different periods, the blank control group and control group the OD value of serum were increased, but there is no obvious difference in comparison within groups (P>0.05), moxibustion serum groups in different time slots within group comparisons, osteoblast OD570value were significantly increased.Conclusion1Mild moxibustion can obviously improve the kidney yang deficiency in postmenopausal osteoporosis patients clinical symptoms, relief of pain in osteoporosis, and is superior to other treatment group.2Mild moxibustion can improve of osteoporosis with lumbar pain around the site and the infrared skin temperature value, effectively improve the kidney yang deficiency syndrome.3Mild moxibustion can improve bone formation related indexes, decreased bone resorption related indicators, and bone turnover the kidney yang deficiency in postmenopausal osteoporosis patients.4Moxibustion serum can promote the proliferation of osteoblasts, can reduce the TNF alpha induced apoptosis of osteoblastic cells number.更多还原