摘要
肿瘤的形成是一个多阶段的缓慢演进过程,免疫是机体最重要的抗癌机制,抗原提呈细胞(antigen-presenting cells,APC)在体内的免疫应答过程中处于极其关键的环节。树突状细胞(Dendritic cells,DC)是目前所知提呈抗原能力最强的免疫细胞,这些功能的发挥都与其成熟密切相关,而DC的成熟是在体内不同组织间的迁移过程中完成的,其中趋化因子及其受体的相互作用是趋动DC迁移的决定因素,DC自身表达趋化因子受体是其从外周组织向淋巴结移动的关键动力。
本研究以树突状细胞为切入点,实验研究对肺瘤平膏按药物组成的功能功效进行拆方,从微观角度比较了不同功效中药对DC的迁移、DC刺激T细胞增殖以及DC-LPAK细胞杀伤肿瘤细胞能力的影响,揭示不同中药在调节免疫中的作用,进一步阐明肺瘤平膏的作用机制。临床研究从宏观出发,首先对肺癌患者DC亚群的变化进行研究,并初步探讨了DC亚群与中医辨证分型的相关性,同时对肺瘤平膏治疗晚期肺癌的近期及远期疗效进行研究,并比较治疗前后DC亚群的变化,初步探讨肿瘤平膏的疗效与DC亚群变化的关系。
一.肺瘤平膏及其拆方对DC趋化功能的影响
采用transwell小室检测DC在趋化因子白细胞介素8(IL-8)和RANTES作用下趋化指数(CI)的改变。结果发现不同中药对DC的趋化能力有不同的影响。IL-8的作用下,益气组中药DC趋化性最强(CI=1.112),活血组(CI=1.084)、肺瘤平组(CI=1.027)次之,而解毒组DC趋化性最低(CI=0.874),益气组与空白血清组比较,P＜0.05,益气组、活血组与解毒组比较,P＜0.05。在RANTES的作用下,肺瘤平组DC趋化性最强(CI=1.185),依次为活血组(CI=1.075)、益气组(CI=1.048),解毒组DC趋化性最低(CI=0.917),肺瘤平组与空白血清组、解毒组比较,P＜0.05。说明肺瘤平膏、益气药、活血药对DC的迁移有不同促进作用,而解毒药物则抑制DC的迁移,不同类中药对DC的迁移影响不同,在不同的趋化因子的作用下,DC的迁移功能也不相同。
二.肺瘤平膏及其拆方作用下DC表面CCR7、CXCR4的改变
通过realtime-PCR的方法,对DC表面趋化因子受体CCR7、CXCR4的表达进行了分析,以阐明不同治则中药对DC趋化影响的分子机制。结果发现:不同类中药作用下,DC表面趋化因子受体CCR7、CXCR4的表达不同。益气组CCR7表达最高,活血组、肺瘤平组次之,解毒组最低,其中,益气组、活血组与解毒组比较,P＜0.05。而CXCR4,益气组、肺瘤平组表达最高,活血组次之,解毒组最低,肺瘤平组、益气组、解毒组与空白血清组比较,P＜0.01,解毒组与其他各组比较,P＜0.01。提示不同中药作用下DC趋化能力的改变可能与趋化因子受体的表达有关。
三.肺瘤平膏及其拆方调节免疫突触及刺激T细胞增殖作用的动态成像研究
采用双光子激光共聚焦显微镜活细胞动态成像及流式细胞仪检测技术,观察DC与T细胞混合培养后,免疫突触(IS)形成及T细胞增殖的动态过程,进一步探讨肺瘤平膏及拆方调节抗肿瘤免疫作用机制及不同。结果发现在一定的时限内,T细胞的增殖与IS形成呈一定正相关;肺瘤平膏、益气方均可促进IS的形成,延长其结合时间;肺瘤平膏无论DC:T的混合比如何,在调节IS形成、刺激T细胞增殖效应方面,明显优于其拆方各组,P＜0.05或0.01,而且,随时相的延长,其效应更加明显;解毒药则在调节IS形成及调节T细胞增殖方面,和其他各组相比较,均不同程度显示出一定的抑制趋势,P＜0.05或0.01。说明IS的形成在DC刺激T细胞增殖中发挥重要作用,肺瘤平膏及其益气组分能促进IS形成,刺激T细胞增殖。
四.肺瘤平膏及其拆方对DC刺激LPAK抗肿瘤活性的影响
比较肺瘤平膏及各拆方中药对DC影响LPAK细胞杀伤肿瘤细胞活性的不同,探讨不同治则方药对DC-LAPK的作用,以期为指导临床用药提供初步的实验依据。研究发现,LPAK:Tumor(L:T)为10:1或5:1组时,各中药组DC诱导的LPAK细胞,杀伤活性明显高于对照组,P＜0.05。L:T为10:1时,肺瘤平、益气组、活血组与解毒组及各对照组比较,P＜0.05。L:T为5:1时,各中药组与对照组比较,P＜0.05,肺瘤平、益气中药、活血中药对DC诱导LPAK细胞杀伤肿瘤细胞的能力的影响对解毒中药高。组内比较,即L:T(10:1)与L:T(5:1)比较,解毒组、空白DC对照组、T+LPAK组,P＜0.05,余各组比较,P＞0.05。肺瘤平组、益气组、活血组在L:T为5:1与10:1时,诱导LPAK的杀伤活性基本相同,与LPAK细胞的比例关系不大。可见肺瘤平膏、活血药、益气药可不同程度增强DC-LPAK杀伤肿瘤细胞的作用,而解毒药则对其有抑制作用。
五.肺瘤平膏及拆方对肺癌Lewis小鼠脾T细胞增殖能力及CTL的影响
通过比较不同中药对Lewis肺癌小鼠脾淋巴细胞增殖能力及CTL的影响,以阐明肺瘤平膏的免疫机制。结果显示荷瘤各组脾T淋巴细胞增殖能力均较正常组降低,具有显著性差异(P＜0.01),与模型对照组比较,正常对照组和肺瘤平组脾T细胞增殖指数均明显增高,有显著统计学差异,P＜0.01;各组与肺瘤平组比较,同样有统计学差异,P＜0.01、0.05。CD8~+CD28~+(CTL)比例各组间比较无统计学差异,P=0.092,从高到低依次:肺瘤平组、正常对照组,益气组、活血组、CTX组、解毒组、模型对照组。与模型组比较,正常对照组、肺瘤平组P＜0.05;与正常对照组比较,CTX组、解毒组、模型对照组P＜0.05;另外,肺瘤平组与CTX组、解毒组比较,P＜0.05。结果表明肺瘤平膏较其他拆方各组更能刺激脾T淋巴细胞的增殖,并可增加了CTL在淋巴细胞中的比例,可以推测这是肺瘤平膏抗肿瘤的免疫机制之一。
六.非小细胞肺癌患者外周血DC亚群测定及意义
选用99例非小细胞肺癌(NSCLC)患者,并以23例健康人做对照,观察NSCLC患者外周血中DC亚群的变化,探讨其与临床特征的关系,并采用ROC曲线进行特异性和灵敏性检验。结果发现患者外周血树突状细胞DC1(CD11c~+)的比例与对照组比较显著降低(P=0.009),DC2(CD123~+)的比例与对照组比较无明显变化(P=0.655),DC1/DC2较对照组低(P=0.177);DC1、DC1/DC2与患者分期之间比较有显著性差异(P=0.032、0.001),DC1在患者KPS之间比较也有明显差异(P=0.023),KPS＜60患者低于KPS≥60者。ROC曲线分析发现肺癌患者外周血DC1、DC1/DC2比率曲线下面积分别为0.671,0.702(P=0.011、0.003)。DC1/DC2与生存时间有关,生存时间大于1年患者与小于1年患者比较,P=0.028,二分类Logistic分析和Cox分析发现DC1/DC2与肺癌患者的生存时间有关,为肺癌患者生存的独立预后因素(P=0.034、0.024)之一,DC1/DC2高者生存时间长。表明对NSCLC患者进行外周血DC亚群监测,有助于全面了解其免疫状态,判断预后。
七.非小细胞肺癌患者外周血DC亚群与辨证分型的相关性研究
选择99例NSCLC住院患者,探讨DC亚群与中医辨证分型的相关性。研究发现肺癌的辨证分型和患者的性别、年龄、分期、病理类型、KPS以及CEA的正常与否无明显相关性。DC1和DC2在各证型之间基本差异不大,DC1由高到低依次为气虚痰湿证、阴虚热毒证、气虚瘀滞证、气阴两虚证,DC2依次为气虚痰湿证、气阴两虚证、阴虚热毒证、气血瘀滞证。DC1/DC2比值的变化在各证型之间有显著差异,P=0.012,气虚痰湿证比值最高,气阴两虚证比值最低。初步说明DC1/DC2与肺癌中医辨证分型有一定相关性。
八.肺瘤平膏改善非小细胞肺癌患者免疫状态及预后的临床研究
采用随机单盲平行对照的临床研究方法,将符合纳入标准的60例非小细胞肺癌患者随机分为治疗组和对照组,治疗组给予肺瘤平联合化疗,对照组单纯化疗,观察近期疗效、免疫功能及生存时间的变化。结果发现:瘤体疗效,治疗组CR 0例,PR 9例,SD 14例,PD 6例,有效率33.33%,对照组CR 0例,PR 7例,SD 13例,PD 10例,有效率26.67%,两组比较P＞0.05。治疗组患者DC1、DC1/DC2、NK细胞与对照组比较治疗后较前增高(P＜0.05、0.01、0.05)。生存分析发现治疗组1年生存率43%,对照组1年生存率28%,两组的总生存时间比较(x~2=1.764,P=0.184)。说明肺瘤平膏在提高晚期肺癌患者的1年生存率,改善生活质量,延长生存期同时可以明显升高DC1比例和DC1/DC2比值,改善机体免疫功能。
九.DC亚群变化与判断肺癌临床疗效及预后的初步探讨
通过观察肺癌患者治疗后T淋巴细胞亚群及DC亚群与生存期的关系,比较其在评估临床疗效方面的价值。结果发现CD4~+/CD8~+升高者1年生存率38%,降低者生存率36%,差异无统计学意义(x~2=0.100,P=0.752)。DC1/DC2升高者1年生存率50%,降低者生存率24%(x~2=6.769,P=0.009)。以DC1/DC2及生存期做为变量,进行双变量相关分析,Spearman's相关系数为0.302,P=0.019,相关性显著。初步表明DC1/DC2的变化对判断药物干预后肺癌的预后有更高的临床意义,治疗后升高者生存时间长。
结论
实验研究
1.肺瘤平膏、益气中药可促进DC的迁移,解毒中药抑制DC迁移,其机制在于不同中药对DC表面趋化因子受体表达影响不同,益气药促进其表达,解毒药抑制其表达;
2.肺瘤平膏、益气中药均可促进DC与T细胞混合培养后免疫突触(IS)的形成,延长其结合时间,从而促进T细胞增殖,解毒药反之;
3.DC可以明显提高LPAK细胞的杀伤活性,肺瘤平膏、活血中药、益气中药有不同程度协同作用,而解毒药则与之相反。
4.肺瘤平膏能刺激脾T淋巴细胞的增殖,并可增加了CTL在淋巴细胞中的比例,拆方研究显示益气组药物作用较明显。
临床研究
1.肺瘤平膏在提高晚期肺癌患者的1年生存率,改善生活质量,延长生存期,同时可以明显升高DC1比例和DC1/DC2比值,改善机体免疫功能;
2.DC1/DC2与肺癌患者的生存时间有关,为肺癌患者生存的独立预后因素,对肺癌患者外周血DC亚群进行监测,有助于对疾病的预后进行判断;
3.DC1/DC2与中医辨证分型有一定相关性,值得我们进一步研究。
Tumor's formation is a slowly evolution process,in which the immunity is the most important anti-cancer mechanism.Antigen presenting cell(APC) is the key factor in immune response process in vivo.Dendritic cell(DC) is so far the strongest immune cell which has the ability to present antigen.The exertion of its function has close relationship with its maturity.DC's maturity completes with its immigration in different tissues.The interaction between Chemokine and Chemokine receptor is determinative factor to moves the DC migration,to adjust the DC circulation.The Chemokine receptor of DC is essential power for its migration from peripheral constitution to lymph node.
We take DC as breakthrough point,try to reveal the sense of Strengthening Body Resistance and Consolidating Constitution theory through experimental study and clinical research.By experimental study,we compare the difference of DC maturity,DC migration and function bring by FLP and its decomposed recipes analysis(DRA) groups,to prove the function to adjust immunity by Benefiting vital energy medicine.Through clinical research,we discovered immune function with nonsmall-cell lung cancer(NSCLC) patients usually is in inhibitory state,and the change of DC subset is one of its performance,which is correlate with the TCM syndrome types.The clinical value is about prognosis.The FeiLiuPing Extract(FLP) can raise 1 year survival rate,lengthen live time.
1.Influence of FLP & DRA on DC chemotactic function
We use transwell to examine CI change of DC under action of IL-8 & RANTES and find that different TCM has different influence on DC chemotactic function. Under the action of IL-8,invigorating Qi herbs strengthens DC chemotactic function(CI=1.112),blood activating herbs(1.084),FLP(1.027),disintoxicating herbs is the lowest(0.874).Compare with YiQi group & HuoXue group,P＜0.05. Under the action of RANTES,FLP group get the highest CI(1.185),HuoXue group (1.075),YiQi group(1.048),JieDu group(0.917).Compare with FLP group,P＜0.01. The result is different kind of TCM has different influence on DC chemotactic function.With different chemotactic factor,it shows different effects,invigorating Qi herbs strengthens DC chemotactic function,while disintoxicating herbs inhibits DC chemotactic function..
2.DC surface CCR7、CXCR4 change under the action of FLP&DRA
Through realtime-PCR method,the expression of chemotactic factor receptor CCR7,CXCR4 on DC surface was analyzed to clarify the molecular mechanism that different TCM changes chemotactic activity of DC.Results show that the expression of CCR7 in YiQi Group was the highest,while HuoXue Group,FLP Group lower, JieDu Group is the lowest.Comparing with YiQi Group,HuoXue Group & JieDu group,P＜0.05.The expression of CXCR4 in YiQi Group & FLP Group was highest, HuoXue Group is lower,JieDu Group is the lowest.Comparing with Blank Serum group and FLP group,YiQi Group,JieDu Group,P＜0.01.The results show that different TCM has differet inflence on chemotactic factor receptor of DC Surface. This is also one of the mechanism that with different chemotactic factor,different TCM has different action on DC chemotactic function.
3.the dynamic imaging of T cells proliferation and regulation of Immunologic synapse(IS) with FLP and DRA
To observe dynamic imaging about living cells including DC and T cells after culturing compounded Using two-photon laser scanning confocal microscope and flow cytometry technology.The immunological synapse(IS) and process of T cell proliferation were observed to discuss the regulating mechanism of anti-tumor immunity.The results showed that mutual migration and accumulation were observed 30 min later when mature DC and T cells were mixed in vitro,observed that the formation of IS 60 min later,after 120 min-1 Oh,with the IS formation,the T cells Obviously proliferation,mutual aggregation phenomenon intensified.And IS reduced after 20h,with gradually gathered more uniform distribution,in a certain time limit and the proliferation of T cells was certainly positive correlation to IS. FLP and invigorating Qi herbs may promote the formation of IS,extend the time of combination.FLP is superior to its DRA in regulating IS formation and proliferation effect of T cells,P＜0.05 or 0.01.The effects were more obvious with time extension. Compared to other groups,JieDu group inhibited it to a extent,P＜0.05 or 0.01.All results show the formation of IS play an important role in the the proliferation of T cells stimulated by DC.FLP can enhance DC-antigen-presenting function,and promote IS formation,so as to stimulate T cell proliferation.
4.FLP & DRA's inflence on DC stimulating LPAK anti-tumor activity
Comparing FLP & DRA's different influence on DC sitmlating LPAK anti-tumor acitivity and dicussing the different action of DC-LARK under different TCM,provides an initial experimental basis on clinical medication.Research found that the killing activity is significantly higher than comparision group when LPAK: Tumor(L:T) eaquals 10:1 or 5:1,all P＜0.05.When L:T eaquals 10:1,FLP group, compare YiQi Group,HuoXue Group and JieDu group with comparision group,all P＜0.05.When L:T eaquals 5:1,compare all TCM group with comparision group,all P＜0.05.FLP,invigorating Qi herbs,blood activating herbs has higher effects than disintoxicating herbs.Comparing within groups,i.e.comparing L:T(10:1) with L:T(5:1),JieDu Group,blank DC group,PL group,P＜0.05.The balanced group, P＞0.05.FLP group,YiQi Group & HuoXue Group has similar effects on stimulating LPAK's killing acitivity,while L:T eaquals 10:1 or 5:1.This shows that FLP,blood activating herbs & invigorating Qi herbs can strengthen the action of DC stimulating DC-LPAK of killing tumor cells to different extent,however,disintoxicating herbs has little inhibition action.
5.The CTL and proliferative capacity of T cell in spleen of Lewis lung cancer with FLP and DRA
To compare CTL and proliferative capacity of T cell in spleen of Lewis lung cancer with FLP and DRA.The results showed that T-lymphocyte proliferation of all bearing-tumor groups were lower than the normal group,P＜0.01.FLP group was lower than normal group,but higer than others,P＜0.05.The rate of CTL to CD8~+T in FLP group and CTX group were both rising,compared to the model group, P＞0.05.we concluded that FLP could enhanced proliferative capacity of T cell and increased the rate of CTL,which were one of its immune mechanism to resist cancer.
6 Sense to determinate DC subset in Peripheral blood of NSCLC patients 99 NSCLC patients were chosen to compare with 23 healthy volunteer.To observe the changes of NSCLC patients's Peripheral blood DC subsets and discuss its relationship with clinic features,ROC curve was adopted to compare its specificity and sensitivity.Results show that the patients's DC1 percentage is significantly lower than comparision group(P=0.009);DC2 has no obvious change(P=0.655),DC1/DC2(P=0.177);DC1,DC1/DC2 has significant difference(P=0.032,0.001),DC1 has obvious difference between different KPS(P=0.023).To analyse ROC curve of DC1,DC1/DC2 ratio in Peripheral blood of Lung cancer patients,the area under the curve is 0.671,0.702(P=0.011,0.003). DC1/DC2 has relationship with survival time.Patients with more than 1 year survival time compare with less than one year patients,P=0.028.Both Logistic and Cox analysis show that DC1/DC2 has relationship with Lung cancer patients's survival time,which is Lung Cancer patients's independent prognostic factor.(P=0.034,0.024).This shows that monitoring Peripheral blood DC subsets of NSCLC patient is conducive to a comprehensive understanding of its immunization status and predicting the prognosis.
7.Relevant research on Peripheral blood DC subsets of NSCLC patients & with the TCM syndrome types
99 NSCLC patients were chosen to study the relevance of DC subsets and the TCM syndrome types.Research found that the TCM syndrome types of lung cancer has no obvious relevance to patient's sex,age,pathology as well as KPS & CEA. DC1 & DC2 has no great difference between each type.Decending sequence of DC1 is:deficiency of vital energy and phlegmatic hygrosis、yin asthenia and pyretic toxicity、energy-stagnation and blood stasis、deficiency of both vital energy and yin. and DC2 was:deficiency of vital energy and phlegmatic hygrosis,deficiency of both vital energy and yin、yin asthenia and pyretic toxicity、energy-stagnation and blood stasis.DC1/DC2 ratio value have obvious difference in each type,P=0.012, deficiency of vital energy and phlegmatic hygrosis is the highest,deficiency of both vital energy and yin is the lowest.The Results of survival analysis on Lung cancer patiens of different types shows that only yin asthenia and pyretic toxicity has a lower survival rate.The other types has no obvious changes,deficiency of vital energy and phlegmatic hygrosis is a little higher.With Log-Rank analysis,x~2=6.051, P=0.109.This shows that DC1/DC2 has some relevance to TCM syndrome types of Lung cancer.
8.Clinic research on NSCLC patient's immune status & prognosis with FLP
60 NSCLC patients was divided into two groups randomly,30 in treatment group and 30 comparision group.Patients in threatment group were treated with FLP plus chemotherapy,while the comparision group only with chemotherapy.In the mealtime,to observe the changes of recent curative effects,immue function and survival period.Results show that curative effects is:Treatment group has CR 0 case, PR 9 cases,SD 14 cases,PD 6cases,the effective percentage is 33.33%,while the comparision group has CR Ocases,SD 13cases,PD 10cases,the effective percentage is 26.67%,P＞0.05.Patients of treatment group has higher DC1,DC1/DC2,NK cells comparing with the comparision group(P＜0.05,0.01,0.05seperately).Survival analysis shows that one year survival rate of treatment group is 43%,while the comparision group is 28%.Comparision of total survival period of the two groups (x~2=1.764,P=0.184).This show that FLP plus themotherapy can obviouly promote the proliferation of NK cells,adjust the change of DC subsets,improve immune function,hence increase the one year survival rate of lung cancer patients,improve life quality and prolong survival.
9.Comparative study of relations between curative effect & prognosis of patients with lung cancer and T lymphocyte subsets & DC subsets
Observing the ralationship between lung cancer patient's post-treatment T lymphocyte & DC subsets and survival period,assess the value of clinic curative effets.Results show that patients with rising CD4+/CD8+ have 38%one-year survial rate,on the contrary,has a survival rate of 36%,the difference have no statistic significance(x~2=0.100,P=0.752).Patients with rising DC1/DC2 have a 50% one-year survival rate,on the contrary,24%(x~2=6.769,P=0.009).With DC1/DC1 plus survival period as variables,we do bivariate relevance analysis,the Spearman's correlation coefficient is 0.302,P=0.019,which means a significant relevance.This shows that changing of DC1/DC2 has much more clinic significance to the estimation of lung cancer prognosis.Those patients who has a rising rate after treatment will survive longer.
Conclusion
Emperiment study
1.FLP,invigorating Qi herbs and blood activating herbs can promote DC migration,disintoxicating herbs inhibit DC migration,the mechanism is that different herbs has different influence on the expression of chemotactic factor receptor on DC surface.
2.FLP & invigorating Qi herbs can both promote the formation of IS(the mixed culture of DC & T cell),prolong its combination period,thus to promote the proliferation of T cells.Disintoxicating herbs shows a certain inhibition trend.
3.DC could obviously increase LPAK cell's killing activity to tumor cells.FLP, blood activating herbs and invigorating Qi herbs has synergism to some extent, whereas disintoxicating herbs has the contrary effects.
4.FLP can enhanced proliferative capacity of T cell and increased the rate of CTL,which were one of its immune mechanism to resist cancer.
Clinical study
1.FLP can raise lung cancer patient's one-year survival rate,improve life quality,prolong survival period and improve immune function,which DC1 an d DC1/DC2 ratio were raised.
2.DC1/DC2 is related with survival time,and is the independent prognos tic factor for the survival of patients with lung cancer.It is helpful for prognos is to monitor DC subsets of NSCLC patients.
3.DC1/DC2 is in relevance with TCM syndrome types.We should invest igate further.
引文
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