温热与卡铂联合对胃腺癌（SGC—7901）细胞的作用

本实验以体外培养人胃腺癌细胞为实验模型，选择卡铂与４０℃，４２℃联合应用，采用先药后热、药药同时，先热后药３种方案对细胞的生长抑制及杀伤规律进行了观察。表明：１。温热与ＣＢＤＣＡ对ＳＧＣ－７９０１细胞均有一定的抑制增殖和直接杀伤作用；温热与药物联合应用对细胞具有协同抑制增殖和杀伤作用；温热与药物联合应用对细胞具有协同抑制增殖和杀伤作用。     

热疗和顺铂对放射治疗人卵巢癌细胞的增强作用研究

目的观察热疗和顺铂对放射治疗的增强作用,以探索一种临床更为有效的肿瘤治疗方法。方法采用Ｘ线照射或后的人卵巢癌细胞Ａ２７８０－Ｓ（对顺铂敏感）和Ａ２７８０－ＣＰ（抗顺铂）用顺铂和热疗处理,观察两者对亚致死剂量辐射损伤修复（ＳＬＤＲ）的抑制作用。结果热疗（４０℃,１小时）或顺铂处理１小时均不能抑制这一修复,但两者联合应用则对两个细胞系均有明显效果。ＳＬＤＲ的受抑程度取决于处理的时机。对于抗顺铂的Ａ２７     

外源性p53基因增加卵巢癌细胞对顺铂的敏感性

 目的 探讨外源性ｐ５３基因转染对人卵巢癌细胞株的化疗敏感性的影响。方法用脂质体介导的转染技术，将人野生型ｐ５３基因的真核表达载体导入不表达ｐ５３的卵巢癌ＳＫＯＶ－３细胞中，经Ｇ４１８筛选，Ｎｏｒｔｈｅｒｎｂｌｏｔ及Ｗｅｓｔｅｒｎｂｌｏｔ鉴定后，观察其对顺铂作用后的 ＳＫＯＶ－３细胞的集落形成及凋亡的影响。结果 外源性Ｐ５３基因在转染细胞中有效表达，并增强了顺铂对ＳＫＯＶ－３细胞集落形成的抑制作用及促进了顺铂诱导的细胞凋亡。结论 外源性ｐ５３基因能增加卵巢癌细胞对顺铂的敏感性，两者联合作用能更大程度地杀灭肿瘤细胞。     

SPC－A－1／DDP耐药细胞株的建立及其生物学特性的初步观察

目的建立耐药细胞株，观察其生物学特性。方法采用体外诱变和药压的方法，建立耐顺铂（ＤＤＰ）的人肺腺癌细胞株ＳＰＣ－Ａ－１的耐药株（ＳＰＣ－Ａ－１／ＤＤＰ）。结果ＳＰＣ－Ａ－１／ＤＤＦ耐药细胞株能耐受的ＤＤＰ浓度为耐ＤＤＰ细胞株的１．５～１５倍，且细胞增殖和传代及耐药性均甚稳定。其倍增时间为３４．３５±０．３５３６ｈ，细胞被抑制５０％时的药物浓度（ＩＣ５０）为４５μｇ／ｍｌ，耐药指数为５．６２５，集落形成率为５３．９７％±２．４９３２％。结论ＳＰＣ－Ａ－１／ＤＤＰ耐药株可以作为研究抗癌药物耐药性机制的实验模型。     

人肺腺癌细胞经化疗药物作用后对LAK细胞的敏感性

在体外实验中研究了８种化疗药物作用后的个旧人肺腺癌细胞（ＧＬＣ－８２）对ＬＡＫ细胞的敏感性。ＧＬＣ－８２细胞本身对化疗药物不敏感，经药物作用２４小时后用３Ｈ－ＴｄＲ释放法检测ＬＡＫ细胞对其杀伤率。结果表明：顺铂（ＣＤＤＰ）、卡铂（Ｃａｒｂｏ）、阿霉素（ＡＤＭ）、丝裂霉素（ＭＭＣ）及长春新硷（ＶＣＲ）处理后的ＧＬＣ－８２细胞对ＬＡＫ细胞的敏感性增强；环磷酰胺（ＣＴＸ）、足叶乙甙（ＶＰ１６）及５－氟脲嘧啶（５－Ｆｕ）则无明显影响。实验提示：对化疗不敏感的肺腺癌用ＣＤＤＰ、Ｃａｒｂｏ、ＡＤＭ、ＭＭＣ及ＶＣＲ后加用ＬＡＫ细胞可提高肺癌治疗的有效率。     

温热与卡铂联合对胃腺癌（SGC－7901）细胞的作用

本实验以体外培养人胃腺癌（ＳＧＣ－７９０１）细胞为实验模型，选择卡铂（ＣＢＤＣＡ）与温热４０℃、４２℃联合应用，采用先药后热、热药同时、先热后药３种方案，对细胞的生长抑制及杀伤规律进行了观察。表明：１．温热与ＣＢＤＣＡ对ＳＧＣ－７９０１细胞均有一定的抑制增殖和直接杀伤作用；温热与药物联合应用对细胞具有协同抑制增殖和杀伤作用。２．温热和ＣＢＤＣＡ的联合对ＳＧＣ－７９０１细胞的杀伤和生长抑制作用均较温热或药物单独作用强，但序贯方面以热药同时为佳。３．温热４Ｏ℃作用后细胞“爆发性生长”，表明单纯加热低于４２℃是不适宜的。     

RV/HSV_1-tk/GCV基因治疗系统对卵巢癌细胞体外作用

［目的］观察逆转录病毒载体／单纯疱疹病毒胸腺嘧啶核苷激酶基因／羟甲基无环鸟苷（ＲＶ／ＨＳＶ１－ｔｋ／ＧＣＶ）基因治疗系统对卵巢癌细胞的体外作用及其“旁观者效应”,并探索提高携有ＨＳＶ１－ｔｋ基因的假型逆转录病毒对卵巢癌细胞的感染率的有效途径。［方法］①以ＶＰＣ／ＨＳＶ１－ｔｋｃ细胞培养液的滤过液重复多次感染Ａ０细胞,然后一组以Ｇ４１８筛选阳性克隆,另一组以ＧＣＶ作杀伤试验。②按不同比例分别将ＡＯ／ＨＳＶ１－ｔｋｃ及ＶＰＣ／ＨＳＶ１－ｔｋｃ与ＡＯ细胞混合培养后加入ＧＣＶ, ５天后消化计数。［结果］①随着ＲＶ感染次数增多,所获得的Ｇ４１８筛选阳性克隆数也明显增多,同时,ＧＣＶ对ＡＯ细胞的杀伤效应也有所提高。②在 ＡＯ细胞群体中如 ＨＳＶ１－ｔｋｃ阳性细胞占 ８０％,则 ＧＣＶ对细胞群体的抑制率可达９５．２％。③当 ＡＯ与 ＶＰＣ／ＨＳＶ１－ｔｋｃ以１：４混合培养时, ＧＣＶ对细胞的生长抑制率为９６．６２％。［结论］ ＧＣＶ杀伤ＡＯ／ＨＳＶ１－ｔｋｃ细胞时对ＡＯ细胞具有一定的“旁观者效应”;重复ＲＶ感染及与ＶＰＣ／ＨＳＶ１－ｔｈｃ细胞混合培养可以明显提高ＲＶ／ＨＳＶ１－ｔｋｃ／ＧＣＶ系统对卵巢癌细胞的作用效果。     

抗人肺腺鳞癌单克隆抗体的研制

将人肺腺癌细胞ＳＰＣ、Ａ５４９、Ａ４２７序贯免疫ＢＡＬＢ／Ｃ小鼠，取脾细胞与小鼠骨髓瘤细胞ＳＰ２／０融合。获得一株稳定分泌抗肺腺鳞癌单克隆抗体的杂交瘤细胞株──Ｓ５Ａ１０─２，制备了小鼠腹水抗体并进行了纯化，腹水效价为１．０２×１０７，亚型测定为ＩｇＧ１．免疫组化分析ｓ５Ａ１０－２单抗与肺腺、鳞癌和肺鳞癌有较好的选择反应性，与正常组织无交叉反应，ＷｅｓｔｅｒｎＢｌｏｔ测定Ｓ５Ａｌ０─２在ＳＰＣ细胞膜上的抗原分子量为４０Ｋｄ、５０Ｋｄ和８０Ｋｄ，在Ａ５４９，细胞膜上的抗原分子量为４３Ｋｄ，而与Ａ４２７细胞膜蛋白无明显反应。该抗体的亲和常数为８．４３×１０１０Ｍ－１，Ｓ５Ａ１０－２杂交瘤细胞株，经液氮冻存后复苏，其分泌抗体能力不受影响。     

抗人胰腺癌单克隆抗体介导LAK细胞体外体内抗胰腺癌作用的研究

体外４小时５１Ｃｒ释放试验表明，抗人胰腺癌单克隆抗体（ＹＰＣ３ＭｃＡｂ）能增强ＬＡＫ细胞对Ｃａｐａｎ－２人胰腺癌细胞的杀伤作用，这种ＬＡＫ细胞的抗体依赖性细胞介导的细胞毒作用（ＡＤＣＣ），随ＹＰＣ３ＭｃＡｂ的浓度增加而加强，５０μｇ／ｍｌ的ＹＰＣ３ＭｃＡｂ增加ＬＡＫ细胞的杀伤率约６０％。而对照抗体无此作用。裸鼠体内实验表明，ＹＰＣ３ＭｃＡｂ和ＬＡＫ细胞同时使用，能完全阻止Ｃａｐａｎ－２细胞的生长，优于ＬＡＫ细胞、脾细胞和ＹＰＣ３ＭｃＡｂ的分别作用。结果提示，ＹＰＣ３ＭｃＡｂ和ＬＡＫ细胞的联合应用，对胰腺癌治疗有一定价值。     

肺腺癌细胞系耐药及凋亡相关基因的表达和逆转

目的 探讨顺铂耐药肺腺癌细胞系Ａ５４９＾ＤＤＰ的耐药,凋亡相关基因的表达及其与亲代细胞Ａ５４９的差异,观察差异表达基因的反义寡核苷酸的逆转作用。方法 将人工合成的反义,正义硫代脱氧寡核苷酸（Ｓ－ｏｌｉｇｏｄｌｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,Ｓ－ＯＤＮ）经脂质体包后转染Ａ５４９＾ＤＤＰ细胞,应用ＲＴ－ＰＣＲ检测耐药及凋亡相关基因ｍＲＮＡ水平,免疫细胞化学及流式细胞－抗体检测相应蛋白及增殖抗原Ｋｉ－６７     

Inhibition of Colony Formation of Drug-resistant Human Tumor Cell Lines by Combinations of Interleukin-2-activated Killer Cells and Antitumor Drugs

The cytotoxicity of interleukin-2-activated killer (LAK) cells with or without anticancer drugs against cell lines with acquired drug resistance was evaluated in vitro by colony assay. Human non-small cell lung cancer cell lines, PC-9 and PC-14, human leukemia cell line, K-562, and their sublines resistant to cisplatin (CDDP), PC-9/CDDP and PC-14/CDDP, and to adriamycin (ADM), K-562/ADM, were used as target cells. PC-9/CDDP demonstrated a marked increase in susceptibility to killing by both peripheral blood lymphocytes (PBL) and LAK cells, as compared to the parental cell line, PC-9. The cytotoxicity of PBL and LAK cells against PC-14/CDDP and K-562/ADM was similar to that against their parental cell lines. Moreover, the combination of LAK and CDDP had a synergistic effect on PC-14 and PC-14/CDDP.     

Inhibition of colony formation of drug-resistant human tumor cell lines by combinations of interleukin-2-activated killer cells and antitumor drugs

The cytotoxicity of interleukin-2-activated killer (LAK) cells with or without anticancer drugs against cell lines with acquired drug resistance was evaluated in vitro by colony assay. Human non-small cell lung cancer cell lines, PC-9 and PC-14, human leukemia cell line, K-562, and their sublines resistant to cisplatin (CDDP), PC-9/CDDP and PC-14/CDDP, and to adriamycin (ADM), K-562/ADM, were used as target cells. PC-9/CDDP demonstrated a marked increase in susceptibility to killing by both peripheral blood lymphocytes (PBL) and LAK cells, as compared to the parental cell line, PC-9. The cytotoxicity of PBL and LAK cells against PC-14/CDDP and K-562/ADM was similar to that against their parental cell lines. Moreover, the combination of LAK and CDDP had a synergistic effect on PC-14 and PC-14/CDDP.     

Cell-cell adhesion-independent killing due to lymphokine-activated killer cells against glioblastoma cell lines.

Lymphokine-activated killer (LAK) cells can kill several tumor cells. Their killing activity is generally due to cell-cell adhesion. Cell-cell adhesion of the LAK cells to the target cells is essential for LAK lysis. In this report, however, we describe that the LAK cells can also kill the target cells by cell-cell adhesion-independent killing. Killing occurred after the target cells were exposed to the LAK cells. When the LAK cells were added to glioblastoma cell lines T98G and U373MG (which proliferate by adhering to the bottom of a culture flask), the LAK cells killed them by cell-cell adhesion killing within 4 h (early killing). On the other hand, when small numbers of the LAK cells were added, some of the target cells escaped from the early killing. At 4 and 6 h after the adding the LAK cells, when the LAK cells were discarded from the flask by washing with PBS, the escaped cells still adhered and were alive. However, they ultimately died over the next 24-96 h (late killing). The late killing was the cell-cell adhesion-independent killing, because it occurred after the LAK cells were removed. In this killing, numerous granules and vacuoles appeared in the cytoplasm of the cells. The vacuoles enlarged and then the cells died. The cell death was different from apoptosis, because the nucleus was intact until the late stage and no DNA fragment laddering in the degenerated cells was recognized. The vacuoles were stained with acid phosphatase and the cell death was inhibited with 3-methyladenine (an inhibitor of lysosome), suggesting that the late killing may be autophagic cell death due to activated lysosome. Induction of late killing in tumor cells using the LAK cells may become one approach for cancer therapy.     

Alpha-interferon and lymphokine-activated killer cells in hairy cell leukemia

Lymphokine-activated killer (LAK) cell activity generated from peripheral blood was tested in 6 patients with typical hairy cell leukemia, 3 not on treatment with alpha-interferon (alpha-IFN) and 3 receiving therapy. In all cases, substantial killing of the LAK-sensitive target Daudi was observed, but hairy cells, whether or not they had been pretreated with alpha-IFN, were uniformly resistant to LAK lysis. The hairy cells were also resistant to LAK cell killing generated from normal peripheral blood mononuclear cells. alpha-IFN added at various times during LAK generation had little or no effect on LAK activity. It is concluded that LAK cells are not important in mediating the beneficial effects of alpha-IFN in hairy cell leukemia.     

Cisplatin induces fas expression in esophageal cancer cell lines and enhanced cytotoxicity in combination with LAK cells

OBJECTIVE: To establish a new therapeutic approach for the treatment of esophageal cancer, we investigated an alternative mechanism of immunotherapy for sensitizing target cells to effector cells. METHODS: Six human esophageal cancer cell lines were used. The expression of Fas antigen on tumor cells was determined by flow cytometry. The cytotoxic effect of cis-dichlorodiammineplatinum (CDDP) and anti-Fas antibody was evaluated using an MTT assay. The cytotoxic activity of LAK cells was measured by a (51)Cr release assay. RESULTS: Five out of six esophageal cancer cell lines expressed Fas antigen at various levels (26.2-61.5%), and Fas expression increased after CDDP treatment. The antitumor effect of anti-Fas antibody on the esophageal cancer cell line and the antitumor effect of LAK cells activated by IL-2 were enhanced by pretreatment with CDDP. After concanamycin A treatment to specifically evaluate Fas-dependent cytotoxicity, LAK cells expressing Fas ligand killed only Fas-positive cells, but not Fas-negative cells. An anti-Fas neutralizing antibody inhibited this cytotoxicity. DNA fragmentation was shown in a cell line that was treated with CDDP and anti-Fas antibody, and also in the targeted esophageal cancer cell line cocultured with LAK cells. CONCLUSION: Our results suggest a potential clinical application of CDDP as a Fas inducer to make esophageal tumors susceptible to Fas antigen and LAK cytotoxicity.     

Membrane-associated Lymphotoxin Expression and Functional Analysis of Lymphokine-activated Killer Cells Derived from Tumor-infiltrating Lymphocytes

The expression of a membrane-associated lymphotoxin molecule (mLT) on lymphokine-activated killer (LAK) cells obtained from 18 patients with malignant tumors and its role in the tumor cell killing mechanisms were investigated. LAK cells from tumor-infiltrating lymphocytes (TIL-LAK cells) were mainly composed of CD3-positive cells, whereas LAK cells from peripheral blood lymphocytes (PBL-LAK cells) were mainly composed of CD16- and CD56-positive cells. However, mLT was found to be expressed on TIL-LAK cells as well as PBL-LAK cells. The degree of mLT expression correlated with the killing activity of LAK cells towards L929 cells (r=0.806, P <0.01, n = 15), but not with that towards Daudi or K562 cells. Although the degree of mLT expression correlated with the amount of secreted lymphotoxin (LT) in the supernatant of LAK cell culture, the secreted LT itself could not account for the tumor cell killing activity of LAK cells. Polyclonal rabbit anti-LT antibody partially inhibited the killing activities of LAK cells towards L929 cells and this inhibition was found in the combination of autologous tumor cells and PBL-LAK cells. These findings suggest the possibility that the mLT-related cytotoxicity is involved in the tumor cell killing mechanisms of TIL-LAK cells as well as PBL-LAK cells.     

Increased therapeutic efficacy induced by tumor necrosis factor alpha combined with platinum complexes and whole-body hyperthermia in rats.

This study examined the effect of a trimodality therapy of the combination of recombinant human tumor necrosis factor alpha (TNF), whole-body hypertheria (WBH), and cis-diamminedichloroplatinum(II) (CDDP) or cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) on a fibrosarcoma and normal tissue in F344 rats. TNF (1 x 10(5) units/kg) increased the antitumor effect of both CDDP (1.5 mg/kg) + WBH (2 h at 41.5 degrees C) and CBDCA (30 mg/kg) + WBH. Tumor growth delay, which was 1.9 days for CDDP + WBH and 2.7 days for CBDCA + WBH (P less than 0.01 compared to control), was significantly increased to 2.9 days with TNF + CDDP + WBH and 5.4 days with TNF + CBDCA + WBH (P less than 0.05). WBH, TNF, CDDP or CBDCA alone, TNF + CDDP, TNF + CBDCA, or TNF + WBH had no significant effect on tumor growth. In contrast, administration of TNF did not enhance the CDDP- or CBDCA-mediated dose limiting normal tissue toxicity. CDDP + WBH-mediated acute renal injury and CBDCA + WBH-mediated acute myelosuppression, as determined by blood urea nitrogen and peripheral blood cell counts, respectively, were not increased with the addition of TNF to either dual modality therapy. Histopathologically, addition of TNF produced no significant alterations in the kidney and the bone marrow as compared to CDDP + WBH or CBDCA + WBH. These data show that TNF enhanced the platinum + WBH-mediated antitumor effect without increasing normal tissue toxicity, suggesting that TNF may increase the therapeutic efficacy of CDDP or CBDCA combined with WBH.     

Combination chemotherapy of paclitaxel followed by nedaplatin for human ovarian cancer

The antitumor activity of a combination of paclitaxel (TXL) followed by nedaplatin (NDP) against SK-OV-3 human ovarian cancer was evaluated. We also compared the antitumor activity of TXL plus NDP with that of TXL plus carboplatin (CBDCA) or TXL plus cisplatin (CDDP). TXL was injected i.v. daily for four days and either NDP, CBDCA or CDDP was injected i.v. once after the TXL treatment, into tumor-bearing mice. The sequential administration of TXL prior to NDP resulted in enhanced inhibition of tumor growth in comparison with either TXL or NDP monotherapy. The combination in TXL plus NDP was synergistic and superior to that of TXL plus CDDP or TXL plus CBDCA therapy. Histological tests demonstrated that the fraction of BrdU-incorporated cells in tumor tissue was significantly inhibited by the combination of TXL with NDP. These results demonstrated the antitumor efficacy of TXL with NDP against human ovarian cancer and suggest the clinical effectiveness of combination of TXL with NDP.     

Lymphokine-activated killer cells can kill target cells not only by early killing but also by late killing,and the late killing level against autotumor cell line

Generally, lymphokine-activated killer (LAK) cells kill target cells early after the LAK cells adhere to them. In this study, we describe that LAK cells can also kill at a later time, such as 24-96 h. LAK cells were generated from a cancer patient and healthy volunteers. As target cells, the patient's autotumor cell line H41 was used. When LAK cells were added to the target cells in a culture well, the LAK cells killed the target cells by cell-cell adhesion within 1-4 h (early killing), but not all cells were killed. The LAK cells were then removed. However, the remaining cells ultimately died 24-96 h later (late killing). The late killing was different from the early killing because numerous granules and vacuoles appeared in the cytoplasm. The late killing was not induced by adding supernatant of the LAK cell culture, suggesting that LAK-target cell contact may be necessary for the killing. The cell injury was inhibited by 3-methyladenine (lysosome inhibitor). It suggests that the vacuoles may be caused by activated lysosome. The patient's LAK cells induced late killing at high levels. There was a high percentage of CD8(+)CD16(+) cells in the peripheral blood lymphocytes (PBL). This subset induced late killing more effectively than the CD8(-)CD16(+) subset. Killing was more conspicuous against H41 than against allogeneic cell line T98G. This type of killing is noteworthy for understanding of killing mechanism of LAK cells.