肝癌5-FU缓释微粒间质化疗的实验及临床研究
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摘要
一、背景和目的：
原发性肝癌(primay hepatic cancer，PHC)是全世界最常见的恶性实体肿瘤之一，也是第三大癌症相关性死亡因素，每年至少有50万新发病例。包括中国东南，台湾，香港，韩国，泰国，新加坡，马来西亚的东南亚地区和热带非洲国家是全球发病率最高的地区，年发病率10-20／10万，且发病率呈上升趋势。大多数肝癌合并有肝硬化，主要的致病因素有病毒性肝炎，酗酒及黄曲霉素的摄入。肝癌患者预后较差，未经治疗的肝癌患者的自然生存时间仅为8．4个月。肝切除术是治疗肝癌的有效方法，然而只有10-20％的肝癌在诊断时具有可切除性。化疗是肝癌治疗重要的辅助手段。但传统的静脉化疗虽然有一定的效果，但全身副作用较大，患者往往难以耐受。因此改变给药途径，即通过局部或区域性给药，能大大提高肿瘤区域内化疗药物的浓度，增加化疗药物对肿瘤细胞的直接细胞毒性作用，并有效地降低了全身副作用。随着抗肿瘤药物缓释剂型的技术改进和对给药途径的观念更新，区域性化疗：即腹腔内化疗、肝动脉灌注化疗、门静脉灌注化疗、间质化疗等方法，日益成为治疗肝癌的有效方法及手段。而间质化疗是当前研究的热点。所谓间质化疗(interstitialchemotherapy，IC)，即将抗癌药制备成具有缓释作用的给药系统，经不同方式植入(注入)肿瘤组织、瘤周组织的间质中或肿瘤切除后的瘤床，从而达到局部组织的持续、高效的药物浓度，同时降低全身的不良反应。
有鉴于此，从上个世纪90年代开始，国外部分学者和吴院士开始进行肝癌间质化疗的研究。在成功进行了肝癌腹腔内化疗、肝动脉灌注化疗、门静脉灌注化疗等方法的基础上，开始间质化疗的实验研究，并且从2003年开始尝试将缓释化疗药物植入肝癌组织、癌周组织中或肝癌切除后的瘤床。
本课题拟通过细胞实验来证实植入用缓释5-FU能持续抑制肝癌细胞的生长，导致肝癌细胞的凋亡，并且5-FU是以时间依赖性方式抑制肝癌，所以进一步充实植入用缓释5-FU应用于临床的理论依据。并且通过300例肝癌患者，按随机数字表分为5-FU缓释微粒植入组和对照组。植入组150例在肝癌切除术中同时植入缓释5-FU微粒600mg，随访其术后的复发率以及生存率来具体评估肝癌间质化疗的疗效，以期得到指导肝癌间质化疗的证据。
二、研究方法
(一)MTT(噻唑蓝)法检测植入用缓释5-FU抑制人肝癌细胞生长的实验
1．HepG2肝癌细胞培养
用植入用缓释5-FU100mg放入2ml dulbecco’s modified eagle’s medium(DMEM)培养液中，分别取植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液来培养HepG2肝癌细胞。
2．观察HepG2细胞增殖形态
培养72h后于倒置相差显微镜下拍摄细胞状态。
3．MTT法检测HepG2细胞增殖
培养72h后加入MTT继续孵育，4 h后终止培养。用酶标仪在560 nm波长下测定吸光度A，细胞存活率(％)=(试验孔A值／对照孔A值)×100％。
(二)植入用缓释5-FU诱导肝癌细胞凋亡的实验研究
1．取植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液来培养HepG2肝癌细胞72h。采用Hochest染色试剂盒检测细胞凋亡。
2．用缓释5-FU浸泡第0d，第1d，第7d，第14d，第21d和第28d的培养液培养HepG2细胞72h后，用Annexin-V-FITC和PI染色后，流式细胞仪检测细胞凋亡。
(三)肝癌行肝脏切除术中植入缓释5-FU的临床试验
300例肝癌患者，按随机数字表分为5-FU缓释微粒植入组和对照组。植入组在肝癌切除术中将缓释5-FU微粒600mg植入肝创面。全部患者术后均定期随访。
三、结果：
(一)MTT(tetrazolium salt,噻唑蓝)法检测植入用缓释5-FU抑制人肝癌细胞生长的实验
1．HepG2细胞经植入用缓释5-FU处理，可见部分细胞变圆、变钝，细胞体积变小，伴核固缩，细胞由贴壁脱落，悬浮于培养液中，尤以浸泡21天和28天后的处理24h为甚；而对照组则细胞成片生长，饱满舒展，贴壁并少有脱落。
2．MTT检测结果显示，植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液处理HepG2细胞72h后，细胞存活率分别为99．5％±8．2％、78．3％±6．2％、43．2％±4．5％、26．3％±3．2％、14．7％±2．2％和11．6％±1．5％。植入用缓释5-FU以时间依赖性方式抑制肝癌细胞生长。
(二)植入用缓释5-FU诱导肝癌细胞凋亡的实验研究
1．用Hochest染色观察到随着缓释5-FU释放时间的延长，凋亡细胞数目也随之增加，其变化趋势同MTT的结果基本一致。
2．流式细胞仪检测结果提示，随着缓释5-FU释放时间的延长，早期凋亡细胞数依次为0％、1．19％、2．26％、3．69％、4．92％和17．92％；呈逐渐升高趋势，坏死和晚期凋亡细胞数依次为0．10％、1．33％、3．77％、4．95％、6．27％和9．35％；总凋亡数依次为0．10％、2．52％、6．03％、8．64％、11．17％和27．27％，也呈逐渐升高趋势。
(三)肝癌行肝脏切除术中植入缓释5-FU的临床试验
植入组术后2、3年肿瘤复发率分别为14．0％和23．3％，与对照组23．3％和34．7％比较(0．01 四、结论：
(一)MTT检测结果显示，植入用缓释5-FU可以在28天内持续释放，并且缓释5-FU以时间依赖性方式抑制肝癌细胞生长。
(二)Hochest染色从形态学角度直观地观察到缓释5-FU以时间依赖性方式导致HepG2细胞的凋亡增加。
(三)流式细胞仪检测到缓释5-FU随着释放时间的延长对HepG2细胞凋亡比例增加。
(四)肝癌行肝切除术中植入缓释5-FU不良反应轻微，安全、简便、有效，在一定程度上能有效地降低术后局部复发率，提高治疗效果，能延长肝癌患者的生存期。
Experimental and Clinical Research on Sustained-release 5-FU Microparticle Interstitial Chemotherapy of Primary Hepatic Cancer
BACKGROUND & OBJECTIVE:
Primay hepatic cancer (PHC) is a disease that occurs when the tumor originates in the liver and did not spread from another organ. It is now one of the most common solid tumor in the world and the third leading cause of cancer-related death. More than 500 000 new cases are currently diagnosed yearly, with an age-adjusted worldwide incidence of 10-20 per 100 000 population and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. In Asia and Africa, hepatocellular cancer is the most common type of malignancy and frequently develops in patients who have liver cirrhosis. Unfortunately, PHC carries a dismal prognosis due to rapid growth rate and nonspecific clinical presentation during the early stages of the disease . The spontaneous survival time of the untreated group is only 8.4 months. Surgical resection is considered to be the curative treatment of patients with PHC. However, only 10-20% of PHC are found to be resectable at the time of diagnosis. In addition recurrence of PHC is frequent even after curative surgical treatment. Chemotherapy holds great theoretical appeal in treating this disease, and systemic chemotherapy has been shown to prolong survival in some patients with PHC . However, orthodox intravenous chemotherapy is also not widely tolerated,because it is effective in some patients but its side effect is relatively more. Thus, the development of effective chemotherapy methods is urgently needed for the control of this disease. Interstitial chemotherapy (IC) is a relatively new concept postulated in last 80's. IC is a relative targeting therapy through which therapeutic agents are locally released from a drug carrying system for an extended period of time while maintaining low systemic drug composure. The drug releasing product could be administered through implantation or injection into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes. The high local drug concentration is particularly useful for killing the surgical inoperable tumors, invisible tumors, isolated tumors cells, and the micro-metastases in lymph nodes.
From 1990's, some researchers, including my renowned adviser prof. Wu Mengchao advanced the new concept on interstitial chemotherapy. Li W-X et al. observed the effectiveness and safety of interstitially implanted 5-FU in treating 139 patients with esophageal cancer, and found that the one year recurrence rate after surgical resection was 38.30% (18/47)、23.36%(10/43) and 14.29% (7/49) , respectively, in patients treated with surgical resection alone, plus implanted 5-FU (200-400mg) and plus implanted 5-FU (400-800mg) . 800mg 5-FU was still well tolerated. Therefore, the authors believe that interstitial chemotherapy, which could provide the needed area with high drug concentration for long period of time with less systemic side effects, is expected to be a potential and promising choice for treating esophageal cancer.
Based on above considerations, Anhui Wuhu Zhongren Pharmaceuticals Co., LTD has developed novel drug sustained releasing implants for treating primay hepatic cancer, including sustained release 5-fluorouracil (ZHONG REN FU AN) . This product could increase drug concentration in tumor local areas by up to thousand folds for up to 4 weeks with only trace amount of systemic exposure. Tumor growth inhibition could be increased by up to 70%-90% as compared to systemic chemotherapy.
Although surgical resection is recognized as the first option for the treatment of primay hepatic cancer, recurrence after tumor resection is the main factor affecting negatively the prognosis of the patients. Residual cells are the major reasons causing recurrence leading poor prognosis. After surgery, the immune function of the hosts declines, with the variety of growth factors being simultaneously secreted, which makes residual cancer cells or precancerous lesions actively grow, therefore highly contributing to early cancer recurrence. Therefore, timely and effectively controlling the residual tumor cells appears to be very important. However, surgical trauma greatly decreases the tolerance of the body to conventional chemotherapy. Since 2003 we started implanting or injecting sustained release 5-fluorouracil into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes.
The purpose of this study is to confirm the sustained-release 5-FU microparticles which can keep on inhibiting hepatoma carcinoma cell and inducing apoptosis by cell experimental , and to get the proof of interstitial chemotherapy of primary hepatic cancer in hepatectomy by clinical research .
METHODS:
1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU and detection the growth of HepG2. by MTT
(1) Culture of the hepatoma carcinoma cell (HepG2)
The hepatoma carcinoma cell (HepG2) was cultured in dulbecco's modified eagle's medium(DMEM) which immersed with sustained-release 5-FU for Od, 1d, 7d, 14d, 21d and 28d.
(2)Detection of the growth of HepG2 by inverted phase contrast microscope.
Detection of the growth of HepG2 by inverted phase contrast microscope after 72h.
(2)detection of the growth of HepG2 by MTT.
Detection of the growth of HepG2 by inverted phase contrast microscope after 72h cultured in MTT.
2. the cell apoptosis induced by sustained-release 5-FU
(1) Hochest staining
Culture of the hepatoma carcinoma cell (HepG2) after Hochest staining and detection of the cell apoptosis .
(2) flow cytometry
Culture of the hepatoma carcinoma cell (HepG2 )and detection of the cell apoptosis by flow cytometry.
3. evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy300 HCC patients underwent hepatectomy from JAN 2003 to JUL 2004. All patients were randomly divided into control group and interstitial chemotherapy group according to random number table. In the control group, 150 patients were treated with hepatectomy alone, whereas in the interstitial chemotherapy group, 150 patients were treated with hepatectomy plus implanting sustained-release 5-FU microparticle on surgical margin. All patients were followed up regularly.
RESULTS:
1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU anddetection the growth of HepG2 by MTT
(1) The hepatoma carcinoma cell (HepG2) with sustained-release 5-FU turned into rounding, blunting, shrinking with karyopyknosis , shedding from the adherence, and suspending in the culture medium in time-dependent manner .But the control group was normal.
(2) The survival rate of cells was determined by MTT assay. The survival rate of 0d, 1d, 7d, 14d, 21d and 28d was 99.5%±8.2%, 78.3%±6.2%, 43.2%±4.5%, 26.3%±3.2%, 14.7%±2.2% and 11.6%±1.5% in the interstitial chemotherapy group, respectively. So 5-FU microparticles could keep on releasing during 28days and inhibiting hepatoma carcinoma cell in time-dependent manner.
2 The cell apoptosis induced by sustained-release 5-FU
(1) The more time the sustained-release 5-FU released, the more cells were induced to apoptosis observated by Hochest staining. So the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.
(2)The 0d,1d, 7d, 14d, 21d and 28d early apoptosis ratio was 0.0%, 1.19%,2.26% , 3.69%, 4.92% and 17.92% in the interstitial chemotherapy group investigated by flow cytometry, respectively. The late apoptosis cell rate was 0.10%,1.33%,3.77% , 4.95%, 6.27% and 9.35%, the total apoptosis ratio was 0.10%,2.52%,6.03% , 8.64%, 11.17% and 27.27%. The sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.
3. Evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy The 2-, 3- and 4-, 5-year recurrent rate was 14.0%,23.3%,32.0% and 43.3% in the interstitial chemotherapy group versus 23.3%,34.7% ,47.3%and 59.3% in the control group, respectively, with a difference in the 2-, 3-year recurrent rates (0.01 CONCLUSIONS:
1.We detected the sustained-release 5-FU microparticles (ZHONG REN FU AN) could keep on releasing during 28days and inhibiting hepatoma carcinoma cell in time-dependent manner by MTT.
2.We detected the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner by Hochest stain.
3.We detected the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner by flow cytometry.
4.Local interstitial chemotherapy using sustained release implants, in contrast, appear to be particularly useful for timely, effectively, and selective controlling the residual cells that lead recurrence. Implanting sustained release drugs into tumor cavity can not only quickly and effectively kill cancer cells but also reduces systemic toxicities associated with systemic chemotherapies. Combining cancer resection with local drug implantation is simple and highly practicable, and is expected to improve cancer prognosis, therefore being a new valuable way for drug administration for treating hepatic cancer.
原发性肝癌(primay hepatic cancer，PHC)是全世界最常见的恶性实体肿瘤之一，也是第三大癌症相关性死亡因素，每年至少有50万新发病例。包括中国东南，台湾，香港，韩国，泰国，新加坡，马来西亚的东南亚地区和热带非洲国家是全球发病率最高的地区，年发病率10-20／10万，且发病率呈上升趋势。大多数肝癌合并有肝硬化，主要的致病因素有病毒性肝炎，酗酒及黄曲霉素的摄入。肝癌患者预后较差，未经治疗的肝癌患者的自然生存时间仅为8．4个月。肝切除术是治疗肝癌的有效方法，然而只有10-20％的肝癌在诊断时具有可切除性。化疗是肝癌治疗重要的辅助手段。但传统的静脉化疗虽然有一定的效果，但全身副作用较大，患者往往难以耐受。因此改变给药途径，即通过局部或区域性给药，能大大提高肿瘤区域内化疗药物的浓度，增加化疗药物对肿瘤细胞的直接细胞毒性作用，并有效地降低了全身副作用。随着抗肿瘤药物缓释剂型的技术改进和对给药途径的观念更新，区域性化疗：即腹腔内化疗、肝动脉灌注化疗、门静脉灌注化疗、间质化疗等方法，日益成为治疗肝癌的有效方法及手段。而间质化疗是当前研究的热点。所谓间质化疗(interstitialchemotherapy，IC)，即将抗癌药制备成具有缓释作用的给药系统，经不同方式植入(注入)肿瘤组织、瘤周组织的间质中或肿瘤切除后的瘤床，从而达到局部组织的持续、高效的药物浓度，同时降低全身的不良反应。
有鉴于此，从上个世纪90年代开始，国外部分学者和吴院士开始进行肝癌间质化疗的研究。在成功进行了肝癌腹腔内化疗、肝动脉灌注化疗、门静脉灌注化疗等方法的基础上，开始间质化疗的实验研究，并且从2003年开始尝试将缓释化疗药物植入肝癌组织、癌周组织中或肝癌切除后的瘤床。
本课题拟通过细胞实验来证实植入用缓释5-FU能持续抑制肝癌细胞的生长，导致肝癌细胞的凋亡，并且5-FU是以时间依赖性方式抑制肝癌，所以进一步充实植入用缓释5-FU应用于临床的理论依据。并且通过300例肝癌患者，按随机数字表分为5-FU缓释微粒植入组和对照组。植入组150例在肝癌切除术中同时植入缓释5-FU微粒600mg，随访其术后的复发率以及生存率来具体评估肝癌间质化疗的疗效，以期得到指导肝癌间质化疗的证据。
二、研究方法
(一)MTT(噻唑蓝)法检测植入用缓释5-FU抑制人肝癌细胞生长的实验
1．HepG2肝癌细胞培养
用植入用缓释5-FU100mg放入2ml dulbecco’s modified eagle’s medium(DMEM)培养液中，分别取植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液来培养HepG2肝癌细胞。
2．观察HepG2细胞增殖形态
培养72h后于倒置相差显微镜下拍摄细胞状态。
3．MTT法检测HepG2细胞增殖
培养72h后加入MTT继续孵育，4 h后终止培养。用酶标仪在560 nm波长下测定吸光度A，细胞存活率(％)=(试验孔A值／对照孔A值)×100％。
(二)植入用缓释5-FU诱导肝癌细胞凋亡的实验研究
1．取植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液来培养HepG2肝癌细胞72h。采用Hochest染色试剂盒检测细胞凋亡。
2．用缓释5-FU浸泡第0d，第1d，第7d，第14d，第21d和第28d的培养液培养HepG2细胞72h后，用Annexin-V-FITC和PI染色后，流式细胞仪检测细胞凋亡。
(三)肝癌行肝脏切除术中植入缓释5-FU的临床试验
300例肝癌患者，按随机数字表分为5-FU缓释微粒植入组和对照组。植入组在肝癌切除术中将缓释5-FU微粒600mg植入肝创面。全部患者术后均定期随访。
三、结果：
(一)MTT(tetrazolium salt,噻唑蓝)法检测植入用缓释5-FU抑制人肝癌细胞生长的实验
1．HepG2细胞经植入用缓释5-FU处理，可见部分细胞变圆、变钝，细胞体积变小，伴核固缩，细胞由贴壁脱落，悬浮于培养液中，尤以浸泡21天和28天后的处理24h为甚；而对照组则细胞成片生长，饱满舒展，贴壁并少有脱落。
2．MTT检测结果显示，植入用缓释5-FU释放后第0d，第1d，第7d，第14d，第21d和第28d的培养液处理HepG2细胞72h后，细胞存活率分别为99．5％±8．2％、78．3％±6．2％、43．2％±4．5％、26．3％±3．2％、14．7％±2．2％和11．6％±1．5％。植入用缓释5-FU以时间依赖性方式抑制肝癌细胞生长。
(二)植入用缓释5-FU诱导肝癌细胞凋亡的实验研究
1．用Hochest染色观察到随着缓释5-FU释放时间的延长，凋亡细胞数目也随之增加，其变化趋势同MTT的结果基本一致。
2．流式细胞仪检测结果提示，随着缓释5-FU释放时间的延长，早期凋亡细胞数依次为0％、1．19％、2．26％、3．69％、4．92％和17．92％；呈逐渐升高趋势，坏死和晚期凋亡细胞数依次为0．10％、1．33％、3．77％、4．95％、6．27％和9．35％；总凋亡数依次为0．10％、2．52％、6．03％、8．64％、11．17％和27．27％，也呈逐渐升高趋势。
(三)肝癌行肝脏切除术中植入缓释5-FU的临床试验
植入组术后2、3年肿瘤复发率分别为14．0％和23．3％，与对照组23．3％和34．7％比较(0．01
(一)MTT检测结果显示，植入用缓释5-FU可以在28天内持续释放，并且缓释5-FU以时间依赖性方式抑制肝癌细胞生长。
(二)Hochest染色从形态学角度直观地观察到缓释5-FU以时间依赖性方式导致HepG2细胞的凋亡增加。
(三)流式细胞仪检测到缓释5-FU随着释放时间的延长对HepG2细胞凋亡比例增加。
(四)肝癌行肝切除术中植入缓释5-FU不良反应轻微，安全、简便、有效，在一定程度上能有效地降低术后局部复发率，提高治疗效果，能延长肝癌患者的生存期。
Experimental and Clinical Research on Sustained-release 5-FU Microparticle Interstitial Chemotherapy of Primary Hepatic Cancer
BACKGROUND & OBJECTIVE:
Primay hepatic cancer (PHC) is a disease that occurs when the tumor originates in the liver and did not spread from another organ. It is now one of the most common solid tumor in the world and the third leading cause of cancer-related death. More than 500 000 new cases are currently diagnosed yearly, with an age-adjusted worldwide incidence of 10-20 per 100 000 population and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. In Asia and Africa, hepatocellular cancer is the most common type of malignancy and frequently develops in patients who have liver cirrhosis. Unfortunately, PHC carries a dismal prognosis due to rapid growth rate and nonspecific clinical presentation during the early stages of the disease . The spontaneous survival time of the untreated group is only 8.4 months. Surgical resection is considered to be the curative treatment of patients with PHC. However, only 10-20% of PHC are found to be resectable at the time of diagnosis. In addition recurrence of PHC is frequent even after curative surgical treatment. Chemotherapy holds great theoretical appeal in treating this disease, and systemic chemotherapy has been shown to prolong survival in some patients with PHC . However, orthodox intravenous chemotherapy is also not widely tolerated,because it is effective in some patients but its side effect is relatively more. Thus, the development of effective chemotherapy methods is urgently needed for the control of this disease. Interstitial chemotherapy (IC) is a relatively new concept postulated in last 80's. IC is a relative targeting therapy through which therapeutic agents are locally released from a drug carrying system for an extended period of time while maintaining low systemic drug composure. The drug releasing product could be administered through implantation or injection into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes. The high local drug concentration is particularly useful for killing the surgical inoperable tumors, invisible tumors, isolated tumors cells, and the micro-metastases in lymph nodes.
From 1990's, some researchers, including my renowned adviser prof. Wu Mengchao advanced the new concept on interstitial chemotherapy. Li W-X et al. observed the effectiveness and safety of interstitially implanted 5-FU in treating 139 patients with esophageal cancer, and found that the one year recurrence rate after surgical resection was 38.30% (18/47)、23.36%(10/43) and 14.29% (7/49) , respectively, in patients treated with surgical resection alone, plus implanted 5-FU (200-400mg) and plus implanted 5-FU (400-800mg) . 800mg 5-FU was still well tolerated. Therefore, the authors believe that interstitial chemotherapy, which could provide the needed area with high drug concentration for long period of time with less systemic side effects, is expected to be a potential and promising choice for treating esophageal cancer.
Based on above considerations, Anhui Wuhu Zhongren Pharmaceuticals Co., LTD has developed novel drug sustained releasing implants for treating primay hepatic cancer, including sustained release 5-fluorouracil (ZHONG REN FU AN) . This product could increase drug concentration in tumor local areas by up to thousand folds for up to 4 weeks with only trace amount of systemic exposure. Tumor growth inhibition could be increased by up to 70%-90% as compared to systemic chemotherapy.
Although surgical resection is recognized as the first option for the treatment of primay hepatic cancer, recurrence after tumor resection is the main factor affecting negatively the prognosis of the patients. Residual cells are the major reasons causing recurrence leading poor prognosis. After surgery, the immune function of the hosts declines, with the variety of growth factors being simultaneously secreted, which makes residual cancer cells or precancerous lesions actively grow, therefore highly contributing to early cancer recurrence. Therefore, timely and effectively controlling the residual tumor cells appears to be very important. However, surgical trauma greatly decreases the tolerance of the body to conventional chemotherapy. Since 2003 we started implanting or injecting sustained release 5-fluorouracil into the needed areas such as tumor mass, tumor cavity or tumor bed (cavity) left by surgical resection, tumor surrounding tissues, and lymph nodes.
The purpose of this study is to confirm the sustained-release 5-FU microparticles which can keep on inhibiting hepatoma carcinoma cell and inducing apoptosis by cell experimental , and to get the proof of interstitial chemotherapy of primary hepatic cancer in hepatectomy by clinical research .
METHODS:
1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU and detection the growth of HepG2. by MTT
(1) Culture of the hepatoma carcinoma cell (HepG2)
The hepatoma carcinoma cell (HepG2) was cultured in dulbecco's modified eagle's medium(DMEM) which immersed with sustained-release 5-FU for Od, 1d, 7d, 14d, 21d and 28d.
(2)Detection of the growth of HepG2 by inverted phase contrast microscope.
Detection of the growth of HepG2 by inverted phase contrast microscope after 72h.
(2)detection of the growth of HepG2 by MTT.
Detection of the growth of HepG2 by inverted phase contrast microscope after 72h cultured in MTT.
2. the cell apoptosis induced by sustained-release 5-FU
(1) Hochest staining
Culture of the hepatoma carcinoma cell (HepG2) after Hochest staining and detection of the cell apoptosis .
(2) flow cytometry
Culture of the hepatoma carcinoma cell (HepG2 )and detection of the cell apoptosis by flow cytometry.
3. evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy300 HCC patients underwent hepatectomy from JAN 2003 to JUL 2004. All patients were randomly divided into control group and interstitial chemotherapy group according to random number table. In the control group, 150 patients were treated with hepatectomy alone, whereas in the interstitial chemotherapy group, 150 patients were treated with hepatectomy plus implanting sustained-release 5-FU microparticle on surgical margin. All patients were followed up regularly.
RESULTS:
1. Culture of the hepatoma carcinoma cell (HepG2) with sustained-release 5-FU anddetection the growth of HepG2 by MTT
(1) The hepatoma carcinoma cell (HepG2) with sustained-release 5-FU turned into rounding, blunting, shrinking with karyopyknosis , shedding from the adherence, and suspending in the culture medium in time-dependent manner .But the control group was normal.
(2) The survival rate of cells was determined by MTT assay. The survival rate of 0d, 1d, 7d, 14d, 21d and 28d was 99.5%±8.2%, 78.3%±6.2%, 43.2%±4.5%, 26.3%±3.2%, 14.7%±2.2% and 11.6%±1.5% in the interstitial chemotherapy group, respectively. So 5-FU microparticles could keep on releasing during 28days and inhibiting hepatoma carcinoma cell in time-dependent manner.
2 The cell apoptosis induced by sustained-release 5-FU
(1) The more time the sustained-release 5-FU released, the more cells were induced to apoptosis observated by Hochest staining. So the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.
(2)The 0d,1d, 7d, 14d, 21d and 28d early apoptosis ratio was 0.0%, 1.19%,2.26% , 3.69%, 4.92% and 17.92% in the interstitial chemotherapy group investigated by flow cytometry, respectively. The late apoptosis cell rate was 0.10%,1.33%,3.77% , 4.95%, 6.27% and 9.35%, the total apoptosis ratio was 0.10%,2.52%,6.03% , 8.64%, 11.17% and 27.27%. The sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner.
3. Evaluation of the efficacy of intraoperative sustained-release 5-FU chemotherapy The 2-, 3- and 4-, 5-year recurrent rate was 14.0%,23.3%,32.0% and 43.3% in the interstitial chemotherapy group versus 23.3%,34.7% ,47.3%and 59.3% in the control group, respectively, with a difference in the 2-, 3-year recurrent rates (0.01
1.We detected the sustained-release 5-FU microparticles (ZHONG REN FU AN) could keep on releasing during 28days and inhibiting hepatoma carcinoma cell in time-dependent manner by MTT.
2.We detected the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner by Hochest stain.
3.We detected the sustained-release 5-FU could keep on inducing hepatoma carcinoma cell to apoptosis in time-dependent manner by flow cytometry.
4.Local interstitial chemotherapy using sustained release implants, in contrast, appear to be particularly useful for timely, effectively, and selective controlling the residual cells that lead recurrence. Implanting sustained release drugs into tumor cavity can not only quickly and effectively kill cancer cells but also reduces systemic toxicities associated with systemic chemotherapies. Combining cancer resection with local drug implantation is simple and highly practicable, and is expected to improve cancer prognosis, therefore being a new valuable way for drug administration for treating hepatic cancer.
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