The basic research for antitumor effect of tumor necrosis facctor (TNF) against malignant brain-tumor

对部分纯化的大鼠肿瘤坏死因子(TNF)进行了体内和体外抗9L大鼠脑肿瘤的实验。静脉注射OK432和脂多糖(LPS)制备含肿瘤坏死因子血清(TNS)。注射TNS可显著延长荷瘤大鼠的存活时间(瘤细胞接种后29.9~12.6天，未治疗组为20.8±4.4天)。在体外实验中，含50%TNS的培养液显著降低9L脑瘤细胞的存活率，在开始培养的第3、5、7天分别下降了57.6%、50.0%和57.0%。TNS对细胞生长也有明显的抑制作用，表明TNS具有细胞抑制作用。通过DEAE-Sephadex A 50批次离子交换和Sephadex G 200柱层析，对TNS进行了部分纯化。四个组分在肿瘤坏死因子敏感的L(S)细胞、肿瘤坏死因子耐药的L(R)细胞和9L脑瘤细胞中进行了测试。TNS组分4对L(S)、L(R)和9L细胞的抑制率分别为37.5%、88.1%和43.2%。而正常大鼠血清组分4的细胞存活率分别为87.5%、87.8%和82.2%。这些结果有力地表明，在OK432和LPS产生的TNS中存在肿瘤坏死因子。重组人肿瘤坏死因子(-hTNF，由Dainippon制药有限公司提供)在体内还与9L脑瘤进行了对比性试验。当9L肿瘤直径达到10 mm时，经静脉或瘤内注射5000单位的-hTNF5000U加或不加淋巴因子激活的杀伤细胞。每只小鼠每隔一周进行精细注射。结果表明，重组人肿瘤坏死因子与LAK细胞联合应用可显著延长LAK细胞的存活时间(P&lt；0.005)。

The effect partially purified rat tumor necrosis factor (TNF) was tested against 9L rat brain tumor both in vivo and in vitro. The TNF-containing serum (TNS) was produced by intravenous injection of OK432 and lipopolysaccharide (LPS). Injection of TNS significantly (p<0.05) prolonged the survival time of brain tumor-bearing rats (29.9 12.6 days after tumor cell inoculation, as compared to 20.8 4.4 days in the untreated group).In the in vitro assay, medium containing 50% TNS significantly decreased the viability of 9L brain tumor cells, by 57.6%, 50.0%, and 57.0% at 3, 5, and 7 days after the beginning of culture, respectively. TNS also displayed significant inhibition of cell growth, indicating a cytostatic effect. To verify TNS activity, TNS was partially purified by means of the DEAE-Sephadex a 50 batch ion exchange method and Sephadex G 200 column chromatography. Four fractions were tested in TNF-sensitive L(S) cells, TNF-resistant L(R) cells, and 9L brain tumor cells. Fraction 4 of TNS demonstrated 37.5%, 88.1%, and 43.2% cell viability against L(S), L(R), and 9L cells, respectively. On the other hand, fraction 4 of normal rat serum showed 87.5%, 87.8%, and 82.2% cell viability, respectively. These results strongly suggest the presence of TNF in the TNS produced by OK432 and LPS.The effect of recombinant human TNF( -hTNF, kindly supplyed by Dainippon Pharmaceutical Co, LTD.) was also tseted against 9l brain tumor in vivo. When the 9L tumor reached to the 10mm in diameter, 5000unit of -hTNF with or without lymphokine activated killer cell was injected intraven ously or intratumorally. Fine injections were given to each mouse one week interval. The result was that the -htnf with LAK cells siginificantly prolonged their survival time (P<0.005) compared to other groups.