凉血化瘀方治疗中心性渗出性脉络膜视网膜病变临床观察及实验研究

【作者】 李学晶；

【导师】 唐由之；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2009， 博士

【摘要】 目的通过临床病例,观察中药凉血化瘀方治疗中心性渗出性脉络膜视网膜病变(centralexudative chorioretinophy,CEC)的疗效。进一步通过动物实验,观察凉血化瘀中药对脉络膜新生血管(choroidal neovascularization,CNV)的影响,并初步探讨其作用机理,为中药凉血化瘀方用于临床治疗CEC提供依据。方法一、临床病例观察:对70例70眼确诊的中心性渗出性脉络膜视网膜病变患者服凉血化瘀方中药治疗,将70眼分为中药组46眼,联合组15眼,陈旧组9眼。服中药3个月,总观察时间9个月。观察比较治疗前后最佳矫正视力(best corrected vision acuity,BCVA)、荧光眼底血管造影(fundus fluorescein angiography,FFA)和光学相干断层扫描(Optic coherenttomography,OCT)图像。二、动物实验研究:1.建立CNV动物模型,并评价可行性。棕色挪威(brown Norway,BN)大鼠32只一眼行氪激光(659nm)眼底光凝,另一眼作空白对照。激光功率、光凝斑直径和曝光时间分别为360mW、50μm及0.05s。光凝20点。分别在光凝后7,14,21,28d随机选取7只大鼠行眼底照像、FFA检查。随机选取1只大鼠行脉络膜血管铺片检查。眼球标本行组织病理切片光镜、Ⅷ免疫组织化学染色观察。2.评价不同浓度凉血化瘀中药对氪激光诱导BN大鼠CNV形态的影响,从中筛选最佳浓度。659nm激光诱导BN大鼠CNV模型同1,随机分成空白对照组、高浓度、中浓度和低浓度中药组4组,每组6只,每日不同浓度中药灌胃。四组分别在光凝后7、14、21d随机选取2只大鼠行眼底照像、FFA检查。眼球标本行组织病理切片光镜、Ⅷ免疫组织化学染色观察。3.比较中浓度凉血化瘀中药与曲胺耐德(triamcinolone acetonide,TA)对氪激光诱导BN大鼠CNV形态的影响。659nm激光诱导BN大鼠CNV模型同1,随机分成空白对照组(16只),中药组(16只),曲胺耐德组(16只)。空白对照组每日生理盐水灌胃,中药组每日中药灌胃,曲胺耐德组术后第2d玻璃体腔内注射5μL(0.2mg)曲胺耐德。在光凝后7,14,21,28d每组随机选取3只大鼠行荧光素眼底血管造影(FFA)检查,检查后处死,取眼球行HE染色,Ⅷ因子相关抗原免疫组织化学检测;同时,每组选取1只大鼠做脉络膜血管铺片检查。4.评价凉血化瘀中药及TA对氪激光诱导BN大鼠CNV中基质金属蛋白酶-9(Matrixmetalloproteinase 9,MMP-9)和基质金属蛋白酶抑制剂(tissue inhibitor ofmetalloproteinase 2,TIMP-2)表达的影响。659nm激光诱导BN大鼠CNV模型同1,随机分成空白对照组,中药组和TA组各12只。分别在光凝后7,14,21,28d随机选取3只大鼠处死,摘除眼球,行病理切片及免疫组织化学检测MMP-9及TIMP-2蛋白在CNV组织中的表达。结果一、临床病例观察:末次随访时,中药组46眼中,视力平均提高0.21,较治疗前有统计意义;视力≥4.7者33眼(71.7%);视力提高≥2行的25眼(54.3%),视力波动在1行以内者16只眼(34.8%),视力下降≥2行的5眼(10.9%),较治疗前视力明显提高。病灶直径、病灶面积,中央视网膜厚度与治疗前相比无统计学意义上的改变,黄斑容积较治疗前降低。FFA检查发现:显效(CNV闭合)34眼(73.9%),有效(CNV部分闭合)7眼(15.2%),进步(CNV小部分闭合)4眼(8.7%),无效(CNV扩大)0眼,较治疗前显著改善。OCT检查发现:CNV型别由治疗前活动期38眼(82.6%)、中间期8眼(17.4%),转变为有效(瘢痕期)38眼(82.6%),进步(中间期)6眼(13.1%),无效(活动期)2眼(4.4%),具有统计学意义。联合组15眼中,视力平均提高0.12,视力分布及视力提高的情况较治疗前无明显改善,病灶直径、病灶面积、中央视网膜厚度及黄斑容积与治疗前相比均无统计学意义上的变化。FFA检查发现:显效(CNV闭合)13眼(86.7%),有效(CNV部分闭合)1眼(6.7%),无效(CNV扩大)1眼(6.7%),较治疗前显著改善。OCT检查发现:CNV型别由治疗前活动期7眼(46.7%)、中间期4眼(26.7%)、瘢痕期4眼(26.7%)均可出现,转变为有效(瘢痕期)13眼(86.7%),进步(中间期)1眼(6.7%),无效(活动期)1眼(6.7%),具有统计学意义。陈旧组9眼,治疗后,病人主诉视疲劳的症状减轻,视力平均提高0.11,视力提高≥2行者3眼(33.3%),但是,与治疗前相比无统计学差异。病灶情况与治疗前相比无统计学差异。二、动物实验研究:1.659nm激光(360mW,50μm,0.05s)照射BN大鼠视网膜20点后,7～21d显示FFA晚期荧光素渗漏及光镜下CNV厚度逐渐增加,28d时FFA渗漏和CNV厚度开始减少。2.FFA显示:光凝后7～21d,空白对照组CNV渗漏面积逐渐增大,而中药组渗漏面积增加无明显差异;至21d,中、低浓度中药组渗漏面积明显低于空白对照组和高浓度中药组,以中浓度中药组CNV渗漏面积最小。CNV厚度变化:空白对照组7～2ld逐渐增厚,至21d达到高峰;而中药组至14d CNV厚度达到高峰,14～21d CNV厚度变薄,以中浓度中药组CNV厚度最薄。3.FFA显示:光凝后7～28d,空白对照组CNV渗漏面积逐渐增大,而中药组渗漏面积略增加,TA组瘢痕着色明显。CNV厚度:空白对照组逐渐增加,至21d达高峰,28d略有下降;中药组CNV厚度逐渐增加,至14d达到高峰,21d时明显变薄,28d再次增加;TA组7～14d时CNV逐渐变薄,至14d时最薄,此后CNV再次增厚。Ⅷ因子相关抗原阳性反应物吸光度:空白对照组7～14d时逐渐增加,至14d达到高峰,此后下降;中药组7d时达到高峰,此后下降;TA组亦是14d达到高峰,但峰值较空白对照组低,此后下降。脉络膜血管铺片CNV面积:空白对照组7～28d逐渐增大;中药组7～14d达到高峰,14～21d下降,28d时再次增加;TA组7～14dCNV面积逐渐增加,21,28d时瘢痕明显。4.空白对照组在光凝后7d MMP-9达到高峰,此后逐渐下降;中药组在光凝后14d达到高峰,但峰值低于空白对照组,此后逐渐下降;曲胺奈德组在光凝后7d达到高峰,峰值介于空白对照组和中药组之间,此后逐渐下降,在14～21d出现平台期。空白对照组在光凝后14d TIMP-2达到高峰,此后逐渐下降;中药组在光凝后14d达到高峰,峰值高于空白对照组,此后逐渐下降;曲胺奈德组在光凝后14d达到高峰,峰值介于空白对照组和中药组之间,此后下降。结论一、临床病例观察:中药凉血化瘀方可以促进CEC中CNV闭合,可以促进经过西医治疗后仍有CNV渗漏的CEC患者CNV闭合,可能改善陈旧CEC患者的视疲劳症状。二、动物实验研究:1.氪激光诱导BN大鼠的CNV模型是一种较为理想的模型,FFA及CNV厚度分析是CNV定量研究的有效手段。2.中浓度凉血化瘀中药可以有效抑制氪激光诱导BN大鼠的CNV增生,防止CNV渗漏。3.TA玻璃体腔注射可以明显抑制CNV形成,但后期形成瘢痕明显;凉血化瘀中药灌胃不仅有效抑制CNV增生,而且瘢痕较小。4.在下调MMP-9,上调TIMP-2的表达进而抑制CNV形成方面,凉血化瘀中药的作用强于TA。更多还原

【Abstract】 OBJECTIVETo investigate the therapeutic effect of Chinese medicine of LiangXueHuaYu on central exudative chorioretinopathy(CEC).And to evaluate the changes of choroidal neovasculation (CNV) taking Chinese medicine of LiangXueHuaYu,investigate the possibility of LiangXueHuaYu decoction as a therapeutic agent for experimental CNV induced by Krypton laser in Brown Norway rats,and further to study their mechanism.Through the clinical cases and animal experiment,we hope we can provide the basis for LiangXueHuaYu decoction used in clinic to curing CEC.METHODSClinical cases observation:The clinical data of 70 patients(70 eyes) with CEC who had followed up for 9 months after therapy of Chinese medicine for CNV were analyzed.The cases were divided into three groups:Chinese medicine group(46 eyes),combined treatment group(15 eyes) and the scar group(9 eyes).The changes of the best corrected visual acuity(BCVA),Fundus Fluorescein Angiography(FFA) and Optic coherent tomography(OCT) were observed and analyzed.Animal experiment study:1.To investigate the feasibility of Krypton laser-induced choroidal neovasculation(CNV) model in the Brown Norway rats.One eye of 32 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s),the other eye were as controls.CNV was evaluated by FFA,high molecular weight FITC-Dextran(MW 2×10~6) for high resolution angiography in RPE-choroid-sclera flat mounts, and histopathologic examination is performed in 7,14,21 and 28 days after photocoagulation.2.To evaluate the changes of choroidal neovasculation(CNV) taking diffent concentration of LiangXueHuaYu decoction,and choose the best concentration.Both eyes of 24 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s).The rats were divided into four groups:the control group(6 rats),high concentration traditional Chinese medicine(TCM) group(6 rats),median concentration TCM group(6 rats), low concentration TCM group(6 rats).All rats received supplementation everyday.FFA and histopathologic examination is performed in 7,14 and 21 days after photocoagulation.3.To evaluate and compare if taking LiangXueHuaYu decoction and intravitreal triamcinolone acetonide(TA) inhibit experimental CNV induced by laser trauma in a rat as a model of choroidal neovascular membranes(CNVMs).Both eyes of 48 rats received a series of 20 spots of laser irradiation(659nm,360mW, 50μm,0.05s).The rats were randomly divided into three groups:the control group(16 rats), traditional Chinese medicine(TCM) group(16 rats),TA group(16 rats).The control group received 0.9%physiological saline by gastric gavage,the TCM group were administrated LiangXueHuaYu decoction drugs by gastric gavage,and TA group underwent an intravitreous injection with 5uL(0.2mg) of TA.Fundus fluorescein angiography(FFA),histopathologic examination,Ⅷstraining cells by immunohistochemistry and high molecular weight FITC—Dxtran(MW 2×10~6) for high resolution angiography in RPE—choroid—sclera flat mounts were performed on 7,14,21 and 28 days after photocoagulation.4.To evaluate the changes of MMP-9 and TIMP-2 in CNV model after taking LiangXueHuaYu decoction and triamcinolone acetonide(TA).Both eyes of 36 rats received a series of 20 spots of laser irradiation(659nm,360mw,50μm, 0.05s).The rats were divided into three groups:the control group(received 0.9% physiological saline by gastric gavage,12 rats),the TCM group(LiangXueHuaYu decoction drugs by gastric gavage,12 rats) and TA group(intravitreous injection with TA 5μl,0.2mg,12 rats).Histopathologic examination(HE),MMP-9 and TIMP-2 straining by immunohistochemistry were performed on 7,14,21 and 28 days after photocoagulation.RESULTSClinical cases observation:At the end of the follow-up period,in the Chinese medicine group,the BCVA improved 0.21,the BCVA of 33 eyes(71.7%) is higher than 4.7,the vision improved obviously(≥2 lines) in 25eyes(54.3%),kept stable(changed within 1line) in 16 eyes(34.8%),and decreased(≥2 lines) in 5 eyes(10.9%).The results of FFA showed completely closed CNV in 34 eyes(73.9%),partly closed CNV in 7 eyes(15.2%),unclosed CNV in 4 eye(8.7%) and enlarged CNV in 0 eyes.OCT showed scar stage in 38 eyes(82.6%),middle stage in 6 eyes (13.1%) and active stage in 2 eyes(4.4%).In the alignment group,the BCVA improved 0.12, the distribution and the improvement of BCVA,the lesion’s diameter and size in FFA,the central retinal thickness and macular volume in OCT were similar with before treatment.The results of FFA showed completely closed CNV in 13 eyes(86.7%),partly closed CNV in 1 eyes(6.7%),unclosed CNV in 0 eye and enlarged CNV in 1 eyes(6.7%).OCT showed scar stage in 13 eyes(86.7%),middle stage in 1 eyes(6.7%) and active stage in 1 eyes(6.7%).In the scar group,there was no significant improve before and after treatment.Animal experiment study:1.CNV was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21, no significant progress occurred in 14～28 days.The fluorescein leakage and the thickness of laser-induced CNV were increased from day 7 to day 21,were decreased in day 28.2.FFA showed that CNV was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21 in control group,but there was no significant progress occurred in TCM groups.The fluorescein leakage of median and low concentration TCM group were significantly less than high concentration TCM group and the control group on day 21.The variation of CNV in thickness showed that in the control group CNV increases from 7 days to 21 days,and reach the peak on day 21;and in the TCM groups especially in the median concentration TCM group,the CNV increases from 7 days to 14 day,and decreases from 14 days to 21 days.3.FFA showed that in control guoup the fluorescein leakage ingravescence,the fluorescein leakage were less in TCM group than in control group,and in TA group the scar staining were greatly bigger than the control group.Histologically,the CNV membrane was observed beneath the retina and the factorⅧpositive cells were seen.CNV thickness was firstly appeared on day 7 after photocoagulation,reaching the peak on day 21 in control group,and decreased on 28d.In TCM group,CNV thickness was reaching the peak on day 14,and decreased greatly on 21d,increased again on 28d.And in TA group, the CNV thickness was getting thinner from 7 to 14d,get thinnest on 14d,then the CNV thickness was getting thicker.The staining intensity ofⅧin cellular plasma showed that in control group the staining intensity was reaching the peak on day 14,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 7, and then decreased greatly.In TA group the staining intensity was reaching the peak on day 14,but the degree was less than the control group,then decreased greatly. Morphologic and quantitative analysis of flat mounts and histologic sections showed that in control group CNV firstly appeared on day 7 after photocoagulation and peaked in day 28,in TCM group CNV peaked on 14d,and decreased significantly on 21d,then increased on 28d.In TA group,CNV was getting bigger from 7 to 14d,but the scars after photocoagulation were obvious on 21,28d.4.The staining intensity of MMP-9 in CNV showed that in control group the staining intensity was reaching the peak on day 7,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 14,the peak was lower than the control group and then decreased greatly.In TA group the staining intensity was reaching the peak on day 7,but the degree was between the control group and TCM group,then decreased.A platform stage occur on day 14～21.The TIMP-2’s staining intensity showed that in control group the staining intensity was reaching the peak on day 14,and then decreased greatly.In TCM group the staining intensity was reaching the peak on day 14,but the peak was higher than the control group and then decreased greatly.In TA group the staining intensity was reaching the peak on day 14,but the degree was between the control group and the TCM group,then decreased greatly.CONCLUSIONSClinical cases observation:Chinese medicine of liangxuehuayu is an effective treatment for most patients with CNV caused by CEC,can help the close of CNV after western medicine therapy,perhaps can improve the symptoms of old CEC.Animal experiment study:1.The Krypton laser induced model of CNV in the pigment rats may be useful,and the quantitative analysis of FFA and the thickness of CNV are useful for in vivo studies of angiogenesis and its modulation via various therapy.2.The median concentration LiangXueHuaYu decoction can suppress experimental CNV induced by Krypton laser.3.Intravitreal injection of triamcinolone acetonide(TA) can inhibit experimental CNV induced by laser trauma in a rat as a model,although on 28d,the scar was obvious.The LiangXueHuaYu decoction can suppress experimental CNV,and the scar was lesser.4.The LiangXueHuaYu decoction and TA can also suppress experimental CNV induced by Krypton laser through the regulation of MMP-9 and TIMP-2,and the LiangXueHuaYu decoction has a stronger effect to regulate the system of MMPs-TIMPs than TA.更多还原