摘要
研究背景：肝癌在我国肿瘤相关死亡率中仅次于肺癌,位居第二。对于局灶性肝癌无论手术还是微波、射频等热消融技术都可以达到灭活肿瘤的目的。但肝癌术后具有易复发性(5年复发率为50-70%),患者在根治术后远期生存率仍然较低。放疗、化疗等传统治疗对预防肝癌的术后复发没有确切疗效,而免疫治疗以其安全有效、特异性强和无毒副作用等优点,已成为当今基础和临床研究的热点。本研究是在总结前阶段研究的基础上,对免疫治疗程序设计、频次和治疗后评估指标进行改进,并观察患者治疗后免疫指标的变化和临床疗效。
目的：1研究小鼠H22肝癌细胞悬液在现有临床应用的温控条件下(54℃、60℃)微波消融后细胞存活、凋亡和死亡的比例,并观察肿瘤相关抗原表达情况；2研究小鼠原位肝癌经微波消融后不同区域肿瘤相关抗原的表达情况；3研究微波消融联合免疫治疗对患者各项免疫指标和肝功指标的影响；4研究微波消融联合免疫治疗对肝癌患者术后复发、肝外转移及生存的影响。
方法：体外实验：本实验按不同消融温控条件设为3组：对照组,消融温度54℃组和60℃组。消融后两小时流式检测悬液中细胞不同状态百分比,包括存活、凋亡和死亡；免疫荧光检测肿瘤相关抗原表达情况,包括CD147和热休克蛋白70(HSP70)。体内实验：开腹瘤块种植法制作昆明鼠原位肝癌模型8只。肿瘤长至直径约5mm时麻醉开腹,将微波针在直视下插入肿瘤中央,测温针放置在肿瘤边缘,瘤周温控60℃。消融后24小时脱颈处死小鼠,免疫荧光检测肿瘤不同区域(肿瘤中心区、肿瘤边缘区)肿瘤相关抗原(CD147、HSP70)表达情况。
临床研究：1微波消融联合免疫治疗对患者术后免疫和肝功影响的研究：实验组为自愿接受微波消融联合过继免疫治疗的14例肝癌患者,对照组为同期仅行微波消融并自愿进行定期免疫和肝功指标检测的15例患者。两组患者在性别,年龄,肿瘤最大直径及分化程度等一般资料方面没有统计学差异,对两组患者术前及治疗后免疫指标和肝功指标进行比较；同时按自身配对原则对实验组14例患者术前和术后3个月外周血PBMC增殖情况进行比较。2微波消融联合免疫治疗肝细胞肝癌对患者肝内复发,肝外转移和患者生存影响的研究。实验组为自愿接受微波消融联合免疫治疗的14例患者,对照组为单纯进行微波消融治疗的42例患者。两组患者在性别,年龄,肿瘤最大直径及分化程度等一般资料方面没有统计学差异。免疫治疗组患者于微波消融后第9天、第1、3个月各做一个疗程免疫治疗,之后每隔3个月根据患者淋巴细胞绝对计数、T淋巴细胞亚群检测结果综合判断是否需要继续进行免疫治疗。比较两组间肝内复发率,肝外转移率和生存率。
结果：体外研究：流式检测结果,消融后2小时54℃和60℃组细胞存活率低于对照组(P<0.05),凋亡和死亡率高于对照组(P<0.05)。60℃组细胞凋亡率高于54℃组(86.0% vs 61.0%)(P<0.05),存活和死亡率均低于54℃组(P<0.05)。免疫荧光检测结果：两个温控组中均可检测到肿瘤相关抗原(CD147、HSP70),其中60℃组两种抗原表达率均高于54℃组(HSP70：98.33% vs 46.22%,CD147：44.0% vs19.44%)(P<0.05),同一温控组HSP70表达率高于CD147(P<0.05)。体内实验：小鼠原位肝癌微波消融后24小时免疫荧光所见,在肿瘤边缘温控条件为60℃,有CD147和HSP70表达,主要在细胞膜表达,但胞浆和细胞间质部位均可见染色。在肿瘤中心区域温度达85℃时,该区域只有HSP70表达,且在细胞和周围间质区均可见荧光染色,CD147未见表达。
临床研究：1微波消融联合免疫治疗对患者免疫和肝功指标影响的研究：实验组和对照组术前淋巴细胞计数、T淋巴细胞各亚群百分比没有统计学差异(P>0.05),治疗后6个月实验组淋巴细胞计数高于对照组(P<0.05)；实验组细胞毒性淋巴细胞亚群(CD3+/CD8+,CD8+/CD28+,NKT)高于对照组,抑制性T淋巴细胞亚群(CD8+/CD28-,T-reg)低于对照组(P<0.05),表明免疫治疗对患者免疫状态具有正向调节作用；实验组与对照组术前白蛋白和胆碱酯酶比较没有统计学差异(P>0.05),术后6个月实验组高于对照组(P<0.05)；实验组术前和术后3个月抽取45ml外周血分离PBMC计数没有统计学差异(P>0.05),术后3个月PBMC培养终点DC和CIK培养目标亚群比例(CD83+,CD86+,CD3+/CD8+)高于术前,且差异有统计学意义(P<0.05)。2微波消融联合免疫治疗肝细胞肝癌对患者肝内复发,肝外转移和患者生存影响的研究：实验组随访期内(4-26个月,中位随访时间19个月)患者有4例(28.6%)肝内复发,其中包括3例(21.4%)局部肿瘤进展和1例(7.1%)远处复发；对照组随访期内(4-26个月,中位随访时间16个月)有11例(26.2%)肝内复发和1例(2.3%)肝外转移,肝内复发包括2(4.8%)例局部肿瘤进展和9例(21.4%)远处复发。两组间在肝内复发率、局部肿瘤进展率、远处复发率、肝外转移瘤和无瘤生存时间比较无统计学差异(P>0.05)。随访期内对照组有4例患者死亡,实验组没有患者死亡,两组间生存率比较无统计学差异(P>0.05)。
结论：实验研究表明,临床现有温控条件下H22细胞悬液微波消融后,大部分肿瘤细胞死亡或凋亡,并表达肿瘤相关抗原。小鼠原位肝癌微波消融后,肿瘤边缘温度为60℃时可见两种肿瘤相关抗原表达。临床研究表明,微波消融术后联合免疫治疗可以改善患者肝功能,提高淋巴细胞计数,优化T淋巴细胞各亚群比例,并可以提高细胞毒性淋巴细胞的增殖活性。由于实验组例数较少,尚未发现微波联合免疫治疗在预防肝癌术后复发和延长患者生存期等方面的优势,该研究还需进一步扩大病例数,延长随访时间。
Background:Hepatocellular carcinoma (HCC) is a common malignant tumor in China, and the mortality of it is second to lung cancer. For the focal nodular, both surgery and microwave or frequency ablation could eradicate it. However, the high recurrent rate of HCC decreases the patients' long term survival rate even after the tumor had been completely eradicated. So far, most related researches have showed the radiotherapy and chemotherapy had no reliable effect to prevent recurrence, while the immunotherapy has become focus of clinical and experimental study to prevent recurrence which hold multiple advantages like safety, effective, specially targets of tumor cell. In this study, we improved the methods of immunotherapy including procedure, frequency and evolution the reaction after immunotherapy on the basic of our prior study, as well as summarizing the change of immune state and clinical results after immunotherapy.
Objective:1 Research the expression of tumor related antigen of H22 after microwave ablation in cell suspension; 2 Research the expression of tumor related antigen of H22 in different point of ablation zone after microwave ablation of HCC in situ of KunMing mice.3 Study the influence of combination microwave ablation(MWA) with adoptive immunotherapy to the immune state and liver function after MWA of HCC.4 Study the effectiveness of combination MWA with adoptive immunotherapy to decrease the incidence of intrahepatic recurrence and extrahepatic metastasis after MWA of HCC.
Method:In vitro experiment Three groups divided according to varies temperature conditions during MWA in this experiment. The control group, the 54℃group with a temperature condition of 54℃and the 60℃group with a temperature condition of 60℃. Two hours after ablation, the cell states was detected by flow cytometry include death, apoptosis and survival, and then the expression of tumor relative antigen (CD 147, HSP70) were detected by fluorescence in 54℃and 60℃groups. In vivo experiment The orthotopic transplantation tumor model of HCC was established by implanting the small tumor block. The HCC in situ was ablated with laparotomy 11 after implantation. The animal special antenna was used and the output power was 5 W, and the temperature in tumor margin was limited at 60℃. After the mice was cut off the head 24 hours after ablation, the tumor relative antigen were detected by fluorescence in different area of ablation zone (the centre of tumor and the margin of tumor).
Clinical study 1 Clinical research for immunity reaction Fourteen cases were included in experimental group who underwent combination MWA of HCC with immunotherapy and another 15 cases were included in control group who underwent only MWA of HCC. The basic conditions in both groups had no significant difference. All patients had regularly undergone immunotherapy testing and liver function testing. Both test results were compared between two groups.2 Clinical research:fourteen cases were included in experimental group who underwent combination MWA with immunotherapy, while 42 cases were included in control group who underwent only MWA therapy. The basic conditions in both groups had no significant difference. At lease 3 courses immunotherapy were applied in each case of 14 cases in experimental group in 9 days,30 days and 90 days after MWA. And then, it was decided according to the results of lymphocyte count and various T lymphocyte subgroup ratio whether the immunotherapy was continues applied in 6,9,12,18 and 24 months after ablation. The incidences of intrahepatic recurrence including local tumor progression(LTP) and distant recurrence, extrahepatic metastasis and death were compared between two groups.
Results:In vitro experiment According to the results of flow cytometry detection 2 hours after ablation, the survival rates in 54℃group and 60℃group were lower than these in control group(P<0.05) and both apoptosis rate and death rate were higher than these in control group(P<0.05). For same temperature condition, the apoptosis rate in 60℃group is higher than the one in 54℃group(86.0% vs 61.0%)(P <0.05) and survival and death rates were lower than these in 54℃group(P<0.05). The tumor relative antigens(CD147 and HSP70) expressed in 54℃and 60℃groups 2 hours after ablation. The expression rate of both antigens in 60℃group is higher than the one in 54℃group(HSP70:98.33% vs 46.22%, CD147:44.0% vs 19.44%)(P< 0.05), and for the same temperature condition, the expression rate of HSP70 is higher than the one of CD147(P<0.05).
In vivo experiment Twenty-four hours after ablation of HCC in situ of KunMing mice, the immunofluorescence displayed that a number of HSP70 and CD147 antigen was expressed in the margin of tumor where the temperature was approximately 60℃, however, only a few of HSP70 was expressed, and no CD 147 expressed in centre area of tumor where the temperature was approximately 85℃; Both antigens mainly expressed in cell membrance, a little expressed in cytoplasm and intracellular substance.
Clinical study 1 Clinical research for immunity reaction The lymphocyte count and various T lymphocyte subgroups results had no significant difference preoperationly between two groups(P>0.05), the lymphocyte count and cytotoxicity T lymphocyte subgroup ratio(CD3+/CD8+,CD8+/CD28+,NKT) in experimental group were higher than these in control group 6 months after ablation, and the suppressive T lymphocyte subgroup ratio(CD8+/CD28-,T-reg) was lower than the one in control group(P<0.05). The differences help to deduce the immunotherapy has an up-regulated effect on immune system. The difference of albumin and cholinesterase between both groups have no statistical significance preoperatively(P >0.05), but both in immunotherapy group are higher than that in control group 6 months after ablation (P<0.05). In immunotherapy group, the count of peripheral blood mononuclear cell(PBMC) isolated from 45 ml peripheral blood between preoperation and 3 months after ablation has no significant different (P>0.05), but the count of DC and CIK cultured from PBMC in 3 months after ablation is higher than that in preoperation(P<0.05).2 Clinical research The intrahepatic recurrence encountered in 4 cases(28.6%) in follow-up period(4-26 Months, Medium 19M) which included LTP in 3 cases(21.4%) and distant recurrence in 1 cases(7.1%) in experimental group, while intrahepatic recurrence encountered in 11 cases(26.2%)which included LTP in 2 cases(4.8%) and distant recurrence in 9 cases(21.4%), one extrahepatic metastasis and 1 death encountered in control group. The intrahepatic recurrent rate, the LTP rate, the distant recurrent rate, extrahepatic metastasis rate and survival rate had no significant difference between two groups(P >0.05).
Conclusion:The experimental study has shown the two temperature conditions applied in clinic(54℃,60℃) could induce most of tumor cell apoptosis or death in cell suspension, and the cell still expressed tumor antigen after apoptosis or death. There were tumor antigens expression at margin of tumor where the temperature is approximately 60℃, and a little of tumor antigen expression in center area of tumor after MWA of HCC in situ of KM mice. The clinical study has shown the immunotherapy could improve lymphocyte count of periphery blood and the ratio of cytotoxicity T lymphocyte subgroup, the liver function and the proliferation capacity of cytotoxicity immunocyte, decrease the ratio of suppression T lymphocyte subgroup. Because of the limitation of case number in experimental group, the combination MWA with immunotherapy haven't deceased the incidence of intrahepatic recurrence of HCC. It needs a further study which should include more objects and longer follow-up period to making a definitive conclusion.
引文
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