脾虚型胃癌腹膜转移的生物学特性以及中西医结合治疗的基础与临床研究
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摘要
胃癌是最常见的消化道恶性肿瘤，总体5年生存率不及30％，其临床生物学行为中浸润深度、分化程度等是判断临床病理分期和预后的主要指标，淋巴结转移、腹膜种植转移程度等是临床评价侵袭性和判断预后的重要因素，而PTNM分期则是以肿瘤的浸润深度、淋巴结转移范围为主要指标，能较客观地反映肿瘤的生物学行为。胃癌患者术后腹膜转移的发生率约占50％，是影响预后的重要因素，严重危害人们的生命健康。
中医学理论认为脾虚证具有许多中医征候所共有的模糊的、全身性的、慢性的、虚性的、与正常体质呈渐变关系的病理特点，辨证论治表明，脾虚证往往比病更能反映疾病的本质。
现代医学实验和临床研究表明，胃癌在不同的阶段可出现许多证型，其中脾虚在胃癌形成中可能起着至关重要的作用，与胃癌发生、发展的各阶段病变之间呈等级正相关。进展期胃癌尤其是晚期胃癌，临床上常伴有脾虚证。
研究表明，晚期胃癌尤其是合并脾虚者预后差，术后腹膜种植转移的患者治疗效果不理想。因此，研究中医脾虚证与胃癌患者临床生物学行为的关系，建立一种合适的脾虚型人胃癌腹膜移植动物模型，
中文摘要
模拟临床实际，进行中西医结合治疗，对深入研究其转移机理及防治
具有十分重要的意义。
然而，关于中西医结合治疗脾虚型胃癌腹膜转移的临床与实验研
究尚存在很多问题有待探索。如关于脾虚型胃癌患者的临床生物学特
性的研究，利用化疗合并健脾益气中药、细胞因子诱导的杀伤细胞
(eytokine一indueed killer，CIK)等对运用中西医结合方法建立的脾虚
型人胃癌裸鼠腹膜移植模型进行干预，并探讨共刺激分子B7(CD80、
CD86等)和主要组织相容胜复合体(major histoeompatibility eomplex，
MHC)抗原在阻止胃癌细胞免疫逃逸中的分子机制等方面的研究目前
尚无报道。
为此，本论文拟在采用中西医结合方法建立病症结合的脾虚型人
胃癌裸鼠腹膜移植模型的基础上，运用第三代的新型铂类化合物奥沙
利铂(L一01月护)化疗合并健脾益气中药、新型过继免疫效应细胞CIK
等对其进行干预，检测 hB7一1基因转染对人胃癌OCLjM一ZMD3腹膜移
植瘤细胞株生物学特性以及MHC Ib、HLA一DR抗原表达的影响，并
观察脾虚型胃癌患者的临床生物学特性，以及应用健脾益气中药参茂
扶正注射液对其免疫功能的影响。深入研究人胃癌细胞免疫逃逸的分
子机制，探讨中医脾虚证与胃癌临床生物学行为的关系以及中西医结
合治疗脾虚型胃癌腹膜转移的方法及其作用机理，为进一步临床应用
提供理论基础。
中文摘要
第一部分
脾虚型人胃癌裸鼠腹膜移植模型的建立
及其生物学特性的实验研究
目的:建立一种能够稳定传代的脾虚型人胃癌裸鼠腹膜移植模型，
观察其生长特点、胃肠粘膜形态学、肿瘤病理形态学以及染色体和DNA
含量，并检测该模型的免疫功能等生物学特征，探讨其用于中西医结
合抗胃癌作用研究的可行性。
方法:将Balb/。n咖u裸鼠随机分为对照组、脾虚组和健脾组。首
先将脾虚组和健脾组裸鼠喂饲大黄芒硝合剂(按4:1混合)5天以建立脾
虚模型，健脾组给予参茂扶正注射液3天，其它组裸鼠给予等量冷开水
对照。然后各组分别用人胃癌OCUM一ZMD3高侵袭转移细胞株
(5x106/0 .2ml)腹腔移植并传代。采用病理形态学、免疫学和分子遗
传学等方法，观察脾虚型人胃癌裸鼠腹膜移植模型中肿瘤生长、腹水
出现时间及腹水量，宿主体重、带瘤生存时间等生长特点，肉眼、光
镜和电镜下胃肠粘膜形态学、肿瘤病理形态学特征以及染色体和DNA
含量分析，检测外周血自然杀伤细胞(natural killer cell，NK)活性、红
细胞免疫功能等免疫功能的变化等。
结果:和对照组相比，脾虚组裸鼠一般自喂饲大黄芒硝合剂3天起，
均出现不同程度的便塘、腹泻、弓背、畏冷、脱肛，进食量明显减少，
以后逐渐出现消瘦、皮下脂肪逐渐减少，行动缓慢，披毛散乱无光泽，
四肢发冷，尾静脉瘪陷等脾虚证状。当裸鼠腹腔接种人胃癌
OCI)M一ZMD3细胞后，各组均发生腹膜转移结节，裸鼠移植率100%。
中文摘要
与对照组相比，脾虚组裸鼠具有体重降低、腹膜种植结节生长多、血
性腹水出现时间早、腹水容量大、带瘤生存时间短等生长特点(p<0.01);
胃肠粘膜形态学观察显示，脾虚组胃肠粘膜苍白，腺体萎缩，血管扩
张充血，炎性渗出，透射电镜下胃粘膜细胞出现核固缩，核膜消失，
出现淋巴细胞，肠粘膜微绒毛减少、脱落，细胞器水肿，有内陷腔。
而健脾组均有明显改善(P<0.01);与对照组相比，脾虚组外周血NK
细胞活性、红细胞C3b受体花环率(red blood eell e3b:eeepte::ate，
RBC一C3bRR)明显降低，红细胞免疫复合物花环率(red blood cell IC
reeepter rate，咫C一ICR)明显升高(尸<0.01)。而健脾组上述各项指标
均有明显改善(p<0.01)。提示本实验方法可成功建立脾虚型人胃癌裸
鼠腹膜移植模型，该中西医结合病证模型的脾虚表现主要是消化、免
疫功能明显降低，应用健脾益气中药参茂扶正注射液可明显改善该模
型裸鼠的脾虚证状;另外，肿瘤病理形态
The gastric carcinoma is the most common malignant tumor in the alimentary truct, and its total 5-year-survival rate is under 30 percent. Its clinical stage and prognosis are mainly concerned with the tumor's infiltrated depth and differential degree, the lymph node metastasis and peritoneal metastasis are important factors in evaluating tumor's infiltration and prognosis, and PTNM stage can reflect the gastric carcinoma's clinical biological behaviors better. Peritoneal metastasis is a frequent type of recurrence after surgery and the reason for the poor prognosis of gastric carcinoma.
For Traditional Chinese Medicine theory the spleen asthenia syndrome has many pathological features of Chinese Medicine, such as vague, chronic, asthenial, gradually overall changed characteristic in normal body and it can reflect the illness essence more effectively than that of the disease itself.
The modern medicial study and clinical research indicate that the
spleen asthenia syndrome plays an important role in the gastric carcinoma's formation and progress. The advanced gastric carcinoma is often accompanied by the spleen asthenia syndrome.
Our research indicated that the advance gastric carcinoma, particularly accompanied by the spleen asthenia syndrome, had poor prognosis and the treatment effect was bad. The study on treatment for peritoneal metastasis of human gastric cancer with the spleen asthenia syndrome combinated by TCM and western medicine has many problems which ought to be resolved. There were no study on the establishment of model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome and the effect of L-OHP, Shenqi Fuzheng Injection, CIK cells on it. There were few reports about human B7-1(CD80), MHC expression on the surface of gastric cancer cells. And there were few reports about relationship between the spleen asthenia syndrome of TCM and the biological behavior in patients with gastric carcinoma.
In order to solve these problems, this research was designed to establish a model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome and to explore the feasibility of using this model to study the anti-gastric cancer effect of combination of TCM and western medicine. The effect of L-OHP, Shenqi Fuzheng Injection, CIK cells on it was investigated. Furthermore, the effects to biological characteristics and hB7-1, the major histocompatibility
comples class I, II (MHC I, II) antigen expression on the surface of human gastric carcinoma OCUM-2MD3 cells infected by the recombinant adenovirus(rAd) on the basis of generation of replication-deficient hB7-1 recombinant adenovirus was studied. At the same time, the biological behavior in patients of human gastric cancer with the spleen asthenia syndrome and the effect of Shenqi Fuzheng Injection on their immunological function was investigated too. So the method and mechanism of the treatment for peritoneal metastasis of human gastric cancer with the spleen asthenia syndrome combinated by TCM and western medicine and the theory for its clinical utilization were explored.
Parti The Establishment and Biologic Characteristic of Transplantable Peritoneal Metastasis of Human Gastric Cancer in Nude Mice with the Spleen Asthenia Syndrome
Objective : To establish a model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome. To explore the feasibility of using this model to study the anti-gastric cancer effect of combination of TCM and western medicine.
Methods: The Balb/c nu/nu naked mice were divided into 3 groups, control, the spleen asthenia syndrome group (SAS group) and the spleen tonic group (ST group). The nude mice of SAS group and ST group were
feed the Rhubarb and mirabilite Mixture on days to establish the spleen asthenia syndrome model. The ST group were feed Shenqi Fuzheng Injection 3 days and the others were feed cold boiled water. All of the mice were implanted a
中医学理论认为脾虚证具有许多中医征候所共有的模糊的、全身性的、慢性的、虚性的、与正常体质呈渐变关系的病理特点，辨证论治表明，脾虚证往往比病更能反映疾病的本质。
现代医学实验和临床研究表明，胃癌在不同的阶段可出现许多证型，其中脾虚在胃癌形成中可能起着至关重要的作用，与胃癌发生、发展的各阶段病变之间呈等级正相关。进展期胃癌尤其是晚期胃癌，临床上常伴有脾虚证。
研究表明，晚期胃癌尤其是合并脾虚者预后差，术后腹膜种植转移的患者治疗效果不理想。因此，研究中医脾虚证与胃癌患者临床生物学行为的关系，建立一种合适的脾虚型人胃癌腹膜移植动物模型，
中文摘要
模拟临床实际，进行中西医结合治疗，对深入研究其转移机理及防治
具有十分重要的意义。
然而，关于中西医结合治疗脾虚型胃癌腹膜转移的临床与实验研
究尚存在很多问题有待探索。如关于脾虚型胃癌患者的临床生物学特
性的研究，利用化疗合并健脾益气中药、细胞因子诱导的杀伤细胞
(eytokine一indueed killer，CIK)等对运用中西医结合方法建立的脾虚
型人胃癌裸鼠腹膜移植模型进行干预，并探讨共刺激分子B7(CD80、
CD86等)和主要组织相容胜复合体(major histoeompatibility eomplex，
MHC)抗原在阻止胃癌细胞免疫逃逸中的分子机制等方面的研究目前
尚无报道。
为此，本论文拟在采用中西医结合方法建立病症结合的脾虚型人
胃癌裸鼠腹膜移植模型的基础上，运用第三代的新型铂类化合物奥沙
利铂(L一01月护)化疗合并健脾益气中药、新型过继免疫效应细胞CIK
等对其进行干预，检测 hB7一1基因转染对人胃癌OCLjM一ZMD3腹膜移
植瘤细胞株生物学特性以及MHC Ib、HLA一DR抗原表达的影响，并
观察脾虚型胃癌患者的临床生物学特性，以及应用健脾益气中药参茂
扶正注射液对其免疫功能的影响。深入研究人胃癌细胞免疫逃逸的分
子机制，探讨中医脾虚证与胃癌临床生物学行为的关系以及中西医结
合治疗脾虚型胃癌腹膜转移的方法及其作用机理，为进一步临床应用
提供理论基础。
中文摘要
第一部分
脾虚型人胃癌裸鼠腹膜移植模型的建立
及其生物学特性的实验研究
目的:建立一种能够稳定传代的脾虚型人胃癌裸鼠腹膜移植模型，
观察其生长特点、胃肠粘膜形态学、肿瘤病理形态学以及染色体和DNA
含量，并检测该模型的免疫功能等生物学特征，探讨其用于中西医结
合抗胃癌作用研究的可行性。
方法:将Balb/。n咖u裸鼠随机分为对照组、脾虚组和健脾组。首
先将脾虚组和健脾组裸鼠喂饲大黄芒硝合剂(按4:1混合)5天以建立脾
虚模型，健脾组给予参茂扶正注射液3天，其它组裸鼠给予等量冷开水
对照。然后各组分别用人胃癌OCUM一ZMD3高侵袭转移细胞株
(5x106/0 .2ml)腹腔移植并传代。采用病理形态学、免疫学和分子遗
传学等方法，观察脾虚型人胃癌裸鼠腹膜移植模型中肿瘤生长、腹水
出现时间及腹水量，宿主体重、带瘤生存时间等生长特点，肉眼、光
镜和电镜下胃肠粘膜形态学、肿瘤病理形态学特征以及染色体和DNA
含量分析，检测外周血自然杀伤细胞(natural killer cell，NK)活性、红
细胞免疫功能等免疫功能的变化等。
结果:和对照组相比，脾虚组裸鼠一般自喂饲大黄芒硝合剂3天起，
均出现不同程度的便塘、腹泻、弓背、畏冷、脱肛，进食量明显减少，
以后逐渐出现消瘦、皮下脂肪逐渐减少，行动缓慢，披毛散乱无光泽，
四肢发冷，尾静脉瘪陷等脾虚证状。当裸鼠腹腔接种人胃癌
OCI)M一ZMD3细胞后，各组均发生腹膜转移结节，裸鼠移植率100%。
中文摘要
与对照组相比，脾虚组裸鼠具有体重降低、腹膜种植结节生长多、血
性腹水出现时间早、腹水容量大、带瘤生存时间短等生长特点(p<0.01);
胃肠粘膜形态学观察显示，脾虚组胃肠粘膜苍白，腺体萎缩，血管扩
张充血，炎性渗出，透射电镜下胃粘膜细胞出现核固缩，核膜消失，
出现淋巴细胞，肠粘膜微绒毛减少、脱落，细胞器水肿，有内陷腔。
而健脾组均有明显改善(P<0.01);与对照组相比，脾虚组外周血NK
细胞活性、红细胞C3b受体花环率(red blood eell e3b:eeepte::ate，
RBC一C3bRR)明显降低，红细胞免疫复合物花环率(red blood cell IC
reeepter rate，咫C一ICR)明显升高(尸<0.01)。而健脾组上述各项指标
均有明显改善(p<0.01)。提示本实验方法可成功建立脾虚型人胃癌裸
鼠腹膜移植模型，该中西医结合病证模型的脾虚表现主要是消化、免
疫功能明显降低，应用健脾益气中药参茂扶正注射液可明显改善该模
型裸鼠的脾虚证状;另外，肿瘤病理形态
The gastric carcinoma is the most common malignant tumor in the alimentary truct, and its total 5-year-survival rate is under 30 percent. Its clinical stage and prognosis are mainly concerned with the tumor's infiltrated depth and differential degree, the lymph node metastasis and peritoneal metastasis are important factors in evaluating tumor's infiltration and prognosis, and PTNM stage can reflect the gastric carcinoma's clinical biological behaviors better. Peritoneal metastasis is a frequent type of recurrence after surgery and the reason for the poor prognosis of gastric carcinoma.
For Traditional Chinese Medicine theory the spleen asthenia syndrome has many pathological features of Chinese Medicine, such as vague, chronic, asthenial, gradually overall changed characteristic in normal body and it can reflect the illness essence more effectively than that of the disease itself.
The modern medicial study and clinical research indicate that the
spleen asthenia syndrome plays an important role in the gastric carcinoma's formation and progress. The advanced gastric carcinoma is often accompanied by the spleen asthenia syndrome.
Our research indicated that the advance gastric carcinoma, particularly accompanied by the spleen asthenia syndrome, had poor prognosis and the treatment effect was bad. The study on treatment for peritoneal metastasis of human gastric cancer with the spleen asthenia syndrome combinated by TCM and western medicine has many problems which ought to be resolved. There were no study on the establishment of model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome and the effect of L-OHP, Shenqi Fuzheng Injection, CIK cells on it. There were few reports about human B7-1(CD80), MHC expression on the surface of gastric cancer cells. And there were few reports about relationship between the spleen asthenia syndrome of TCM and the biological behavior in patients with gastric carcinoma.
In order to solve these problems, this research was designed to establish a model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome and to explore the feasibility of using this model to study the anti-gastric cancer effect of combination of TCM and western medicine. The effect of L-OHP, Shenqi Fuzheng Injection, CIK cells on it was investigated. Furthermore, the effects to biological characteristics and hB7-1, the major histocompatibility
comples class I, II (MHC I, II) antigen expression on the surface of human gastric carcinoma OCUM-2MD3 cells infected by the recombinant adenovirus(rAd) on the basis of generation of replication-deficient hB7-1 recombinant adenovirus was studied. At the same time, the biological behavior in patients of human gastric cancer with the spleen asthenia syndrome and the effect of Shenqi Fuzheng Injection on their immunological function was investigated too. So the method and mechanism of the treatment for peritoneal metastasis of human gastric cancer with the spleen asthenia syndrome combinated by TCM and western medicine and the theory for its clinical utilization were explored.
Parti The Establishment and Biologic Characteristic of Transplantable Peritoneal Metastasis of Human Gastric Cancer in Nude Mice with the Spleen Asthenia Syndrome
Objective : To establish a model for transplantable peritoneal metastasis of human gastric cancer in nude mice with the spleen asthenia syndrome. To explore the feasibility of using this model to study the anti-gastric cancer effect of combination of TCM and western medicine.
Methods: The Balb/c nu/nu naked mice were divided into 3 groups, control, the spleen asthenia syndrome group (SAS group) and the spleen tonic group (ST group). The nude mice of SAS group and ST group were
feed the Rhubarb and mirabilite Mixture on days to establish the spleen asthenia syndrome model. The ST group were feed Shenqi Fuzheng Injection 3 days and the others were feed cold boiled water. All of the mice were implanted a
引文
1 陈峻青，王舒宝，齐春莲，等．胃癌外科治疗中的若干问题．中华外科杂志，1991；24：220～223
2 尹光耀，张武宁，许福昌，等．胃康复对脾虚证胃粘膜癌前病变上皮细胞核及线粒体Zn、Cu和DNA的影响．中国中西医结合脾胃杂志，2002；4(8)：221～224
3 蒋民主．脾虚与消化道肿瘤的早期临床意义．附消化道肿瘤114例临床分析．新疆中医药，1998；(3)：7
4 尹光耀，张武宁，李国成，等．胃康复治疗脾虚证胃癌前病变的疗效及其作用机理的研究．中国中西医结合杂志，2000；20(3)：176～179
5 郭峰，田宝华，张乐之．现代红细胞免疫学．上海：上海第二军医大学出版社，2002；60～62
6 孙弼纲，田长经，沈鹰．中医“脾虚”症本质的临床探讨．安徽中医学院学报，1983；(3)：3
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