【作者】 潘明沃；

【导师】 陈志强；

【作者基本信息】 广州中医药大学 ， 中医外科学， 2009， 博士

【摘要】 背景前列腺癌是男性常见的恶性肿瘤之一。在美国其发病率居男性恶性肿瘤之首,在我国其发病率已跃居泌尿系肿瘤的第3位。前列腺癌患者确诊时多数已属晚期,并且几乎所有患者经内分泌治疗一段时期后都转变为雄激素非依赖型前列腺癌,因此成为现代医学的治疗难点。广东省中医院泌尿外科多年来潜心研究前列腺癌,通过中医证候调查,发现D₀期前列腺癌中医辨证多属脾虚证、肾气虚证。D₁期前列腺癌中医辨证多属血瘀证,下焦湿热证。D₂期前列腺癌中医辨证多属脾虚证、血瘀证、下焦湿热证、肾气虚证、阴虚痰热证和肾阴虚热证。D₃前列腺癌中医辨证多属气血两虚证,肾阳虚证。根据证候调查结果,结合现代医学进展,制定了以扶正抑瘤为主法的中西医结合方案,治疗本院住院及门诊前列腺癌病例142例,统计分析总生存率、总体中位生存期、总体无进展生存率、骨转移灶数目、PSA、血细胞分析、QLQ-C30量表、并发症发生率等,观察扶正抑瘤法对前列腺癌的实际临床疗效,随访时间1～50月,观察期间死亡16例,总生存率90.14%,总体中位生存期27.7月,总体无进展中位时间25.2个月。PSA复发时间23.9±19.2个月,PSA倍增时间24.1±20.4个月。QLQ-C30量表标准分统计,症状改善有效率81.0%。潮热症状发生率22.4%,骨质疏松发生率13.8%;乳房肿痛发生率11.7%。其中T3～4NxMlb期110例患者总体无进展生存期24.3个月。去势后PSA最低值0,PSA复发时间20.1±15.2个月,PSA倍增时间22.3±17.4个月。骨转移灶消失4例,缩小、变淡79例,无扩散、无增多18例,出现新的转移灶9例,在提高生存质量、延缓肿瘤转移及进展等方面,显示了中医药良好的前景。同时在临床实践中发现前列腺癌患者的细胞免疫功能均低下,大部分患者的细胞免疫功能在应用扶正抑瘤中西医结合治疗后有一定变化,并呈现提高的趋势,这些结果正是越来越受重视的肿瘤免疫治疗所希望达到的目的,而抗肿瘤免疫中特异性细胞免疫中占主导地位。树突状细胞是人体功能最强大的抗原提呈细胞,是特异性免疫的启动者。既然扶正抑瘤法已经初步体现其在细胞免疫调节中的前景,因此,有必要对其在前列腺癌患者体内树突状细胞的影响进一步探索。目的在既往前列腺癌的病因病机、证候规律研究、中西医结合治疗方案对生存质量及PSA、骨转移灶临床疗效研究以及中西医治疗对前列腺癌患者细胞免疫的研究的基础上,应用分别反映树突状细胞的总体数量和活化能力的分子CD1a和CD83在前列腺癌原发灶病理切片中标记树突状细胞,探讨扶正抑瘤法对前列腺癌患者原发灶树突状细胞的数量及活化的影响。方法本研究采用前瞻性平行对照实验设计,病例来源:2005年1月至2009年1月广东省中医院确诊前列腺癌的住院病人57例,根据病理资料和不同的处理方法分为两组,每组分两个亚组。其中第一组18例患者均取得2次病理标本,其中亚组中西医结合治疗组10例,亚组内分泌治疗组8例,中西医结合治疗组采用“扶正抑瘤”中西结合治疗方案（扶正抑瘤中药+常规内分泌治疗）,内分泌治疗组采用常规内分泌治疗方案,治疗时间根据两次取病理时间间隔7天到105天不等;第二组39例患者仅获得1次病理标本,按照病理活检前作为疑似前列腺癌病例时有否接受扶正抑瘤中药早期干预治疗分为亚组中药治疗组和亚组空白对照组,两组未病理确诊前均未接受内分泌治疗;其中中药治疗组19例,在获得病理诊断前作为疑似病例已经使用扶正抑瘤中药,治疗时间7到25天不等,平均8.7天;空白对照组20例,病理诊断前未使用任何治疗措施。用免疫组化技术Envision两步法染色标记所有前列腺癌患者（包括两个组所有亚组的患者）原发灶病理切片的CD1a阳性细胞及CD83阳性细胞。光镜下观察记录这些被标记的树突状细胞数量,阴性病理切片计数为0。进行统计学分析比较中西医结合治疗组与内分泌治疗组、中药组与空白组、中西医结合组与中药组、中药组与内分泌治疗组的CD1a和CD83阳性树突状细胞的均数。成组均数若符合正态分布时比较用成组t检验,不符合正态分布时比较用非参数检验（Mann-Whitney Test）检验,p≤0.05为差异有显著性。配对均数符合正态分布时用配对t检验,不符合正态分布时比较用非参数检验（Wilcoxon秩和检验）,p≤0.05为差异有显著性,统计运用SPSS13.0软件包。结果1、第一组18例患者,其中中西医结合治疗组10例,内分泌治疗对照组8例,中西医结合组治疗前1例CD1a阴性、1例CD83阴性。内分泌治疗对照组治疗后1例CD1a阴性、1例CD83阴性。中西医结合治疗组治疗后的CD1a阳性树突状细胞数量均比治疗前升高,差异有统计学意义,P＜0.05;内分泌治疗组治疗后的CD1a阳性树突状细胞数量均比治疗前升高,差异有统计学意义,P=0.05。中西医结合治疗组治疗后的CD83阳性树突状细胞数量比治疗前升高,差异有统计学意义P＜0.05,内分泌治疗组治疗后的CD83阳性树突状细胞数量比治疗前略高,差异无统计学意义,P＞0.05;中西医结合治疗组CD1a阳性树突状细胞治疗前后差值高于内分泌治疗组,差异无统计学意义,P＞0.05。中西医结合治疗组CD83阳性树突状细胞治疗前后差值高于内分泌治疗组,差异有统计学意义,P＜0.05。2、第二组患者39例,其中中药治疗组19例,空白对照组20例,中药治疗组1例CD1a阴性;空白对照组3例CD1a阴性、2例CD83阴性。中药治疗组CD1a阳性树突状细胞的数量比空白对照组高,差异有统计学意义,P＜0.05,中药治疗组CD83阳性树突状细胞的数量比空白对照组高,差异有统计学意义,P＜0.05。3、中西医结合治疗组的CD83阳性的树突状细胞数量比中药治疗组高,差异有统计学意义,P＜0.05。中西医结合治疗组的CD1a阳性的树突状细胞数量比中药治疗组高,差异无统计学意义,P＞0.05。中药组CD1a阳性的树突状细胞数量比内分泌治疗组低,差异无统计学意义,P＞0.05;中药组CD83阳性的树突状细胞数量比内分泌治疗组高,差异无统计学意义,P＞0.05。4、各种扶正抑瘤中药方案中,中药针剂+中药汤剂组比单纯中药汤剂组的CD1a、CD83均数高,差异有统计学意义,P＜0.05。5、镜下观察可见空白对照组前列腺癌患者的原发灶CD1a、CD83阳性树突状细胞细胞呈胞浆及细胞核染色,形态欠成熟,伸出伪足不明显,染色较前列腺增生组织浅,数目减少以CD83阳性细胞明显。树突状细胞在癌周间质分布较多,在癌组织中分布较少,一般在癌周淋巴细胞浸润处分布较多。扶正抑瘤中药治疗组治疗后前列腺癌患者的原发灶树突状细胞的数量明显增多,在癌及癌周均有分布,染色较深,树突状细胞亦多分布在癌周较多淋巴细胞浸润处,淋巴细胞浸润较空白组密集,CD83阳性细胞形态较空白对照组成熟,伸出伪足明显,数个叠连,染色较深。结论1、前列腺癌患者原发灶树突状细胞的总体数量较良性前列腺组织减少、活化受抑制。扶正抑瘤中西医治疗方案的应用能在一定治疗时段提高前列腺癌患者原发灶的树突状细胞的总体数量及活化能力。2、中西医结合、中药针剂与汤剂联用等治疗方案在一定治疗时段显示出在提高前列腺患者树突状细胞的总体数量与活化方面的联合效应优势。3、使用扶正抑瘤中药后,前列腺癌原发灶树突状细胞的数量增多和活化能力提高的同时能观察到局部淋巴细胞数量增加,其可能机制是树突状细胞活化后有效提呈肿瘤抗原,激活淋巴细胞,引起淋巴细胞增值,从而启动特异性细胞免疫。更多还原

【Abstract】 BackgroundProstate cancer（PCa） is one of the malignant tumors of man’s reprodu ctive system.In US,the incidence of prostate cancer is listed in the fi rst place of the man’s malignant tumors’.In China,its incidence has be en up rising to the 3^rd place of all the urinary tumors’.Most of the indi viduals were diagnosed to be prostate cancer in their later period of can cer progression.Moreover,nearly all of the patients who had received th e incretion therapy would eventually turn to be the hormone refractory ca ses.This makes it tough for Modern Medicine to cure.Urinary Surgical De partment of Guangdong Provincial Hospital of Chinese Medicine had for lon g concentrated on the study of prostate cancer,and had initially explore d its etiology,pathogenesis and syndrome differentiation.The regular pa tterns of Prostate cancer are as follows:Phase D₀:spleen Qi deficiency, kidney Qi deficiency.Phase D₁:blood stasis,lower energizer damp-heat, kidney Qi deficiency.Phase D₂:spleen Qi deficiency,blood stasis,lowe r energizer damp-heat,kidney Qi deficiency,Yin deficiency and phlegm he at,deficiency heat.Phase D₃:Qi and blood deficiency.The according int egrative treatment protocols according to TCM concept "Fuzhengyiliufa" w hich has enhancive effect of quality of life and postponement of tumor pr ogress and metestasis had later been developed,demonstrated good prospec t in clinical used.Prospective study of 142 available cases of inpatient or outpatient in Guangdong hospital of TCM from Sep.2004 to Mar.2007 h ad been conducted.The treat period was 9 months.Collected information o f Karnofsky table、weight、QLQ-C30 table、I—PSS table、ache ctassificatio n table of WHO、table for research in symptom category of prostate cancer in D period、PSA、blood testosterone、ECT of systemic bones、analysis of blood corpuscle.The remedy group is obviously better than the control group in increasing weight,improving the scale of Karnofsky table,alle viating symptoms such as tiredness、pain、insomnia、appetite losing、p ressing breach、constipation、diarrhea,improving the emotion function a nd health condition in whole;improving the symptom of lower urethra;red ucing the incidence rate of the symptom such as tiredness and fatigue,in somnia,desudation,poor appetite,difficult in urethra,pain in waist an d knee,etc.,reducing the amount of PSA and bone metastasis（P＜0.05）,enh ancing the amount of blood corpuscle such as WBC、HGB、PLT in circulation. In recent clinical practice,cellular immunity function decline was seen in all prostate cancer patients.While majority of patients’ cellular im munity function were seen to be enhanced after the application of "Fuzhen gyiliufa" integrative treatment.This is precisely the same goal that all immunotherapies take hope to achieve.In human immune system,specific c ellular immunity plays a key role in fighting against tumor.While dendri tic cell（DC） is deemed to be the most powerful antigen precenting cells, which is the initiator of specific immunity.However,dendritic cell-bas ed immunotherapies only manifest the transient curative effect in partial PCa patients.Since "Fuzhengyiliufa" has been observed to have effect i n immunity function,its influence in dendritic cell should be further ex plored.PurposesTo observe the infiltration and activation of dendritic cell in prima ry foci of prostate cancer using "Fuzhengyiliufa" integrative treatments.MethodsPerspective research,control trail,57 prostate cancer in-patients w ere intaken in the study.The first group 18 patients,who had obtained p athological specimens before and after treatments respectively,were divi ded into two subgroups,therapeutic group 10 patients,and control group 8 patients.The therapeutic group received "Fuzhengyiliufa" integrative t reatment（"Fuzhengyiliufa" TCM treatments combined with routine incretion therapy）,the control group receieved routine incretion therapy,and the therapeutic period ranged between 7 days to 105 days.The second group 3 9 patients,who had only obtained 1 pathological specimen in each,were d ivided into two subgroups according to whether patients received "Fuzheng yiliufa" TCM treatment or not before their biopsy:therapeutic group 19 pa tients;blank control group 20 patients.The therapeutic group received "Fuzhengyiliufa" TCM treatments,the therapeutic period ranged between 7 to 25 days,8.7 days in average;control group didin’t received any trea tment,then marked the CD1a positive dendritic cell and the CD83 positive dendritic cell which infiltrated in these pathological sections with the immunohistochemistry technique of "Envision" two step staining,and rec orded the quantities of these dendritc cells with microscope.The negativ e ones would be recorded as "0" in quatity.As for statistics analysis, dependent t-test would be used to compare means of dependent groups if th e variance meets Gaussion distribution,if not,non-parametric test（Mann -Whitney Test） would be applied,p＜0.05 has the significance for the diff erence.Pair groups t-test would be used to compare means of pair groups if the variance meets Gaussion distribution,if not,non-parametric test （Wilcoxon Test） would be applied,p＜0.05 has the significance for the dif ference.Statistic analysis software SPSS13.0 would be applied.Results1.The first group 18 patients who had obtained twice pathologic samp les in each,therapeutic subgroup 10 patients,with 1 before treatment sa mple CDla negative and 1 before treatment sample CD83 negative;Control s ubgroup 8 patients,with 1 after treatment sample CD1a negative and 1 aft er treatment sample CD83 negative.The quantity of CD1a positive DC after treatment in both groups elevated compared to the quantity of CD1a befor e treatments,the differences had statistical significance.The quantity of CD83 positive DC after treatment in therapeutic group elevated compare d to the quantity of CD83 positive DC before treatment,the differences h ad statistical significance,P≤0.05.The quantity of CD83 positive DC af ter treatment in control group elevated compared to the quantity of CD83 positive DC before treatment,the differences had no statistical signific ance,P＞0.05.The quantity difference of CD1a positive DC of therapeutic group were more than the ones of control group,the differences had no st atistical significance,P＞0.05.The quantity differences of CD83 positive DC of therapeutic group were more than the ones of control group,the di fferences had statistical significance,P＜0.05.2.The second group 39 patients,who had obtained one pathologic samp le,therapeutic group 19 patients,with 1 sample CD1a negative;blank con trol group 20 patients,with 3 sample CD1a negative and 2 sample CD83 neg ative.The quantity of CD1a positive DC in therapeutic group surpass blan k control group,the difference had statistical significance,P＜0.05.The quantity of CD83 positive DC in therapeutic group surpass blank control group,the difference had statistical significance,P＜0.05.3.Single use of "Fuzhengyiliufa" Chinese Medicine group elevated CD8 3 in quantity compared to blank control group,the differernce had statis tical significance,P＜0.05.4.In descriptive study of comparision of various "Fuzhengyiliufa" tr eatments,it could be seen that the CD1a、CD83 elevating effect of conbine d used of herbal decortions and herbal injections were better than single use of herbal decortion,the differences had statistical significance,P＜0.05.5.It could be observed with microscope in blank control group PCa pa tients’ pathological section that the infiltrated dendritic cell declined in quatity and disfunction in activating.Marked by CD1a and CD83,dendr itic cells were stained in their cytoplasmid and nuclear,appeared immatu re in shape that less like dendritic form.Their staining is lighter than the ones in benign hyperplasia tissues,with prominent decline in CD83. Dendritic cell infiltration is more concentrating in mesenchrymal tissues beside tumor tissue with infiltrated lymphocyte arround.In therapeutic group PCa patients’ pathological sections,infiltrated dendritic cell are much more in quantity.Dendritic cell infiltration is more concentrating, not only in mesenchrymal tissues but also in between the tumor cells.Th ey appeared more mature in dendritic form,staining darker.Moreover,the dendritic cells,marked by CD83,are more mature in shape with more conc entrating infiltrated lymphocytes around. Conclusions1.Infiltrated dendritic cells in prostate cancer primary foci were l ess than the ones in benign hyperplasia tissues in quantity.Their activa tion was inhibited.The application of "Fuzhengyiliufafa" integrative tr eatment protocol could increase the quantity of dendritic cell in prostat e cancer primary foci and greatly enhance their activating ability in cer tain therapeutic period.2.It could be seen that the DC quantity elevating effect and the DC activation reinforcement effect of integrative therapy of Chinese Medicin e and incretion therapy and the combination of herbal decortions and inje ctions had advantage over other single used treatments.3.It could be observed that with the elevated quantity and activatin g ability of dendritic cell,local lymphocytes infiltration became more c oncentrating.The probable mechanism was that activating dendritic cells precent tumor antigens to activate T lymphocytes.That leads to prolifera tion and expansion of T lymphocyte.In that way specific immunity was ini tiated.更多还原