低剂量X线辐射对A549细胞凋亡的适应性反应

目的 研究低剂量X射线辐照对A549细胞凋亡的适应性反应及时间效应,并初步探索适应性效应的可能机制。方法 分别用X射线50 mGy、200 mGy、500 mGy照射A549细胞,间隔3 h、6 h、12 h、24 h、48 h后,用20 Gy的效应剂量照射,检测细胞凋亡;并检测初始剂量和效应剂量间隔照射6 h的细胞周期分布及DNA损伤。20 Gy及0 Gy照射组为对照组。结果 低剂量间隔3 h、6 h、12 h、24 h后再接受20 Gy照射,50 mGy～20 Gy、200 mGy～20 Gy、500 mGy～20 Gy 3组的细胞凋亡率均分别显著低于20 Gy组（P < 0.05）;间隔48 h,50 mGy～20 Gy、200 mGy～20 Gy、500 mGy～20 Gy 3组的细胞凋亡率与20 Gy组无显著性差异。低剂量照射与效应剂量间隔6 h,50 mGy及200 mGy叠加20 Gy效应剂量组,G0/G1期细胞百分数显著低于20 Gy剂量组（P < 0.05）,50 mGy～20 Gy、200 mGy～20 Gy组G2/M期细胞百分数显著降低（P < 0.05）,500 mGy～20 Gy组G0/G1及G2/ M期细胞百分数与20 Gy照射组无统计学差异。与20 Gy组相比,3个低剂量叠加20 Gy组细胞及DNA损伤程度降低,但无统计学意义。结论 低剂量X射线照射能诱导A549细胞凋亡的适应性反应,且与初始剂量和效应剂量间隔的时间有关,适应性效应可能与低剂量X射线引起的细胞周期改变有一定关系。     

LXA4对人白血病细胞K562与HL60膜受体ALX表达的影响

目的比较脂氧素A4受体(lipoxin A4 receptor,ALX)在人白血病细胞K562与HL60细胞的表达情况,研究脂氧素A4(Lipoxin A4,LXA4)对K562与HL60膜受体ALX的表达影响,探索LXA4对K562与HL60细胞作用效应强弱不同的可能因素。方法体外培养人白血病细胞K562与HL60,实验分为空白组和LXA4(50、100、200 nmol/L)处理组。采用免疫荧光技术检测K562和HL60细胞是否表达LXA4受体ALX;用不同浓度LXA4作用24 h后,RT-PCR和Western blotting检测并比较各实验组K562与HL60膜受体ALX的表达变化。结果免疫荧光检测显示,K562与HL60胞膜周围及胞浆内有明显绿色荧光,提示二者表达LXA4受体ALX;RT-PCR和Western blotting检测结果表明:随LXA4作用浓度的增高,K562与HL60细胞膜受体ALX的表达呈增高趋势(P<0.05),组间比较,ALX表达差异无统计学意义(P>0.05);K562与HL60空白组比较即从基础水平比较ALX的表达水平无明显差异(P>0.05)。结论 LXA4可上调K562与HL60膜受体ALX的表达,且LXA4对K562与HL60效应强弱的不同并非ALX基础表达水平不同所导致,可能存在其他影响因素。     

低剂量γ射线对小鼠胸腺细胞和脾细胞周期进程的影响

目的以小鼠胸腺细胞和脾细胞周期进程变化为指标,观察低剂量辐射生物效应的机制.方法小鼠接受低剂量γ射线作用后,用流式细胞仪检测小鼠胸腺细胞和脾细胞DNA含量的变化.结果 50～250 mGy照射组小鼠G0/G1期胸腺细胞百分率明显低于假照射组,50mGy和100mGy照射组小鼠S期胸腺细胞百分率明显高于假照射组,说明低剂量γ射线具有促进胸腺细胞由G0/G1期向S期过度的作用.50～250 mGy照射组小鼠（G2＋M）期细胞百分率明显高于假照射组,说明50～250mGyγ射线可以引起小鼠胸腺细胞G2期阻滞.10～250mGy照射组小鼠胸腺G0/G1期脾细胞百分率明显高于假照射组,而10mGy、75mGy和250mGy照射组脾S期细胞百分率明显低于假照射组,说明低剂量γ射线不仅对脾细胞G0/G1期有阻滞作用,而且能够抑制S期脾细胞的DNA合成.结论低剂量γ射线对小鼠胸腺细胞和脾细胞DNA合成分别具有刺激和抑制作用.     

LAK细胞和HSV1-TKc/GCV基因治疗系统对卵巢癌细胞的体外作用

[目的]对LAK细胞和HSV1-TKc/GCV基因治疗系统对卵巢癌细胞的体外作用进行研究分析。[方法]分别将不同比例的LAK细胞和AO细胞混合培养6h(4︰1,20︰1,100︰1,500︰1),采用LDH释放法测定LAK的杀伤活性;HSV1-TKc+及HSV1-TKc-AO细胞按照不同比例培养60h(15︰85,35︰65,50︰50,75︰25,65︰35,85︰15),后更换15μg/ml GCV培养液,6d后采用LDH释放法测定其杀伤活性。对两组实验的效靶比与杀伤活性进行相关性检验,比较两组的杀伤活性。[结果]LAK细胞与靶细胞的比值与杀伤活性呈线性相关性(r=0.996,P﹤0.01),其杀伤活性随着效靶比的增大而增加;AO/HSV1-TKc+与AO/HSV1-TKc-的细胞比例与HSV1-TKc/GCV的杀伤活性成线性相关(r=0.984,P﹤0.01),其杀伤活性随着比例的增大而增加,LAK与HSV1-TKc/GCV两组比较,差异无统计学意义(P﹥0.05)。[结论]LAK细胞与HSV1-TKc/GCV基因治疗系统对卵巢癌细胞的体外具有一定的杀伤作用,但两者比较差异无统计学意义。     

过继免疫治疗的新希望--CHAK细胞

目的：探讨CHAK细胞体外常规生物学活性。方法：采用血球计数板计算外周血单核细胞(PBMC)、LAK细胞及CHAK细胞三组细胞的增殖能力，MTT法检测各组细胞对K562肿瘤细胞株杀伤能力，免疫组化检测细胞表型。结果：与PBMC和LAK相比，CHAK细胞显示出更强增殖能力和较高的细胞毒活性，且CD16^ 、CD56^ 细胞表型明显高于其它两组。结论：CHAK是一类不同与LAK细胞的新型高效杀伤细胞，可能会在临床抗肿瘤治疗中发挥重要的作用。     

香烟提取物对人体NK细胞和LAK细胞活性影响

目的　了解烟雾对人类自然杀伤细胞 (NK)和杀伤细胞 (LAK)活性的影响。方法　抽取 4 0名不吸烟的健康自愿者外周血分离单个核细胞 (PBMC) ,将每名自愿者的PBMC分为A、B、C、D 4份 ,A、B用于检测NK细胞 ,C、D用于检测LAK细胞 ,其中B、D不加香烟提取物作为正常对照 ,A、C加入香烟提取物一起培养 ,以K5 6 2 (NK敏感细胞 )为靶细胞经噻唑蓝 (MTT)法检测NK细胞活性 ,以HL 6 0为靶细胞 (NK耐受细胞 )检测LAK细胞活性。观察香烟提取物对NK细胞和LAK细胞的影响。结果　A组NK细胞平均活性为 (43 95± 5 5 7) %,B组为 (45 84±5 6 1 ) %,二者差异无统计学意义 (P >0 0 5 ) ,两组NK细胞活性段位分布比较差异有统计学意义 (P <0 0 5 ) ,C组LAK细胞平均活性为 (2 0 0 7± 1 78) %,D组为 (2 4 5 0± 2 1 7) %,二者差异有统计学意义 (P <0 0 5 )。结论　香烟提取物可影响LAK细胞活性和少数个体NK细胞活性。降低机体免疫监视功能和对癌变细胞清除的能力 ,可能是吸烟者癌症高发的主要原因之一。     

CD3AK细胞对膀胱癌细胞的体外杀伤活性研究

目的 探讨CD3AK细胞的某些生物学特征、了解其对膀胱癌细胞的体外杀伤活性。方法 采用抗CD3单抗和IL-2共同刺激健康人外周血单个核细胞诱导出CD3AK细胞，体外动态观察CD3AK细胞的形态特征及增殖能力，并与LAK细胞进行比较；以流式细胞仪测定细胞表型：用MTT法测定CD3AK、LAK细胞对膀胱癌细胞株(124)的杀伤活性。结果 (1)CD3AK细胞增殖能力高于LAK细胞；(2)CD3AK细胞为异质细质细胞群，其细胞类型主要是CD8^ 细胞；(3)在效靶比为10：1及20：1时，培养4d的CD3AK细胞对124细胞的杀伤率为均高于同期培养的LAK细胞对124的杀伤率。结论 (1)CD3AK细胞制备简便，增殖能力强，可以为临床提供充足的过继免疫效应细胞；(2)CD3AK细胞是一明显优于LAK细胞的抗肿瘤效应细胞，对膀胱癌细胞具有较强的杀伤作用。CD3AK细胞在膀胱癌过继免疫治疗中具有重要的临床应用前景。     

CIK细胞的诱导及其对肝癌细胞毒作用的体外研究

目的体外诱导细胞因子诱导的杀伤细胞(CIK),并研究其生物学活性。方法从外周血和脐血分离单个核细胞(PBMC),经过细胞因子诱导、培养并扩增CIK细胞,以LAK作比较,流式细胞仪检测CIK细胞表面标志CD3、CD56。3H-TdR释放法检测CIK的增殖能力,MTT法检测对肝癌细胞系SMMC-7721、Bel-7402,正常胎肝细胞L-02的杀伤活性。结果CIK细胞第二周进入快速增殖期,到第31d扩增倍数超过60倍,CD3+CD56+细胞扩增倍数达800倍以上;CIK对肝癌细胞的杀伤能力明显优于LAK细胞(65%～81%);对正常胎肝细胞的细胞毒作用(<5%)。结论CIK细胞是一种具有很强杀瘤活性的免疫活性细胞,具有临床应用前景。     

低剂量辐射诱导小鼠生精细胞凋亡及p53基因表达的适应性反应

目的研究低剂量X射线照射诱导小鼠睾丸生精细胞凋亡及p53基因表达的适应性反应。方法通过不连续泛影葡胺密度梯度离心法分离睾丸组织不同种类生精细胞，采用碘化丙啶荧光探针标记细胞，流式细胞术检测各类生精细胞凋亡，免疫组化法观察生精细胞p53蛋白表达。结果当1．0、2．0或3．0Gy（攻击剂量，D2）照射前6h给予75mGy（诱导剂量，D1）预照射时明显减轻D2对精原细胞和精母细胞的凋亡损伤作用，而对精子细胞和精子影响不明显。当D2照射前6h给予D1预照射时明最减少精原细胞和精母细胞p53基因表达阳性率，而对精子细胞和精子影响不明显。结论低剂量X射线照射可选择性诱导小鼠睾丸精原细胞和精母细胞凋亡的适应性反应，并与D1和D2间隔时间及D2剂量有关。同时，可诱导精原细胞和精母细胞p53基因表达的适应性反应，推测其在低剂量辐射诱导生精细胞凋亡适应性反应分子机制中起重要的调控作用。     

低剂量辐射诱导体外EL-4淋巴瘤细胞周期进程的适应性反应

目的 观察低剂量辐射诱导EL-4淋巴瘤细胞周期进程的适应性反应。方法 用X射线照射离体EL-4淋巴瘤细胞,其诱导剂量(D1)为25~200mGy(12.5mGy/min),攻击剂量(D2)为1.5Gy(287mGy/min),D1和D2间隔6h。通过流式细胞仪检测其细胞周期各时相百分数的变化。结果 当D1和D2分别为25~100mGy和1.5Gy,或分别为75mGy和0.5~2.0Gy,D1+D2各组G₀/G₁期细胞百分数不同程度低于各自D2组,而S期细胞百分数明显高于各自D2组(P<0.05、P<0.01或P<0.001)。结论 上述结果显示,EL-4淋巴瘤细胞在1.0~2.0Gy照射前6h接受25~100mGy照射,可在体外诱导其细胞周期进程的适应性反应。     

硒和维生素E对人白血病周期的影响

方法：应用体外细胞培养法，流式细胞术（ＦＣＭ）研究硒和ＶＥ对人髓系白血病细胞株ＨＬ－６０和非髓系白血病细胞株Ｋ５６２细胞增殖和细胞周期分布的影响。结果：Ｓｅ（５～１１μｍｏｌ／Ｌ）对于ＨＬ－６０，Ｋ５６２细胞的增殖均有显著性抑制作用，ＦＣＭ表明，在Ｓｅ（８μｍｏｌ／Ｌ）作用６～７２ｈ后，ＨＬ－６０和Ｋ５６２细胞分别出现Ｇ１和Ｓ期阻滞现象。ＶＥ在１００～３００μｍｏｌ／Ｌ范围内对ＨＬ－６０细胞具有显著性抑制作用，而在１０～３００μｍｏｌ／Ｌ范围内对Ｋ５６２细胞同样具有显著性抑制作用，且呈剂量相关效应。ＶＥ（１００μｍｏｌ／Ｌ）作用６～７２ｈ后，ＨＬ－６０，Ｋ５６２细胞分别被阻滞于Ｓ期和Ｇ１期。值得注意的是Ｓｅ与ＶＥ对ＨＬ－６０和Ｋ５６２细胞周期分布的影响恰恰相反。结论：硒的抗癌作用和ＶＥ的抗癌作用是互补的，不能互相替代     

Synthesis and induction of G0-G1 phase arrest with apoptosis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one

The multistep synthesis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one 15 has been carried out. The compound showed antiproliferative and apoptotic effects against K562, K562-R (imatinib mesilate resistant), HL60 and multidrug resistant (MDR) HL60 cell lines. Compound 15 showed a pro-apoptotic activity against HL60 and K562 resistant cell lines markedly higher than etoposide and busulfan, respectively. Flow cytometry studies carried out on K562 cells allowed to establish that 15 induces G0-G1 phase arrest followed by apoptosis.     

Low-dose radiation induces adaptive response in normal cells, but not in tumor cells: in vitro and in vivo studies

Biological effects of low-dose radiation (LDR) are distinguishable from those of high-dose radiation. Hormetic and adaptive responses are such two examples. However, whether adaptive response could be induced in tumor cells by LDR, especially under in vivo condition, remains elusive, and was systemically investigated in the present study. Four tumor cell lines: two human leukemia cell lines (erythroleukemia cell line K562, and acute promyelocytic leukemia cell line HL60), and two human solid tumor cell lines (lung carcinoma cell line NCI-H446 and glioma cell line U251), along with one normal cell line (human fibroblast cells, MRC-5), were irradiated with LDR at 75 mGy of X-rays as D1 and then 4 Gy of X-rays as D2 (i.e.: D1 + D2) or only 4 Gy of X-rays (D2 alone). Three tumor-bearing animal models were also used to further define whether LDR induces adaptive response in tumor cells in vivo. Adaptive response was observed only in normal cell line, but not in four tumor cell lines, in response to LDR, showing a resistance to subsequent D2-induced cell growth inhibition. Three tumor-bearing mouse models with U251, NCI-H446 or S180 tumor cells were used to confirm that pre-exposure of tumor-bearing mice to D1 did not induce the resistance of tumor cells in vivo to D2-induced tumor growth inhibition. Furthermore, a higher apoptotic effect, along with higher expression of apoptosis-related genes P53 and Bax and lower expression of anti-apoptosis gene Bcl-2, was found in tumor cells of the tumor-bearing mice exposed to D1 + D2 than those in the tumor cells of the tumor-bearing mice exposed to D2 alone. These results suggest that LDR does not induce adaptive response in the tumor cells under both in vitro and in vivo conditions, which is a very important, clinic-relevant phenomenon.     

Low dose radiation increased the therapeutic efficacy of cyclophosphamide on S(180) sarcoma bearing mice

We examined whether low dose radiation (LDR) exposure (75 mGy) could increase the therapeutic efficacy of cyclophosphamide (CTX) by comparing the effects of tumor suppression, tumor cell apoptosis, cell cycle and proliferation of bone marrow in vivo. Kunming mice implanted with S(180) sarcoma cells were given 75 mGy whole body gamma-ray radiation exposure and CTX (300 mg/kg) by intraperitoneal injection 36 hours after LDR. Proliferation of bone marrow and tumor cells was analyzed by flow cytometry. Cytochrome c leakage from the tumor was measured by Western-blot. We discovered that tumor growth was significantly reduced in the group exposed to CTX add to LDR. The apoptosis of tumor cells increased significantly after LDR. The tumor cells were arrested in G(1) phase in the groups treated with CTX and CTX + LDR, but cell cycle was more significantly arrested in mice exposed to LDR followed by CTX than in mice exposed only to LDR or CTX chemotherapy. Concentration of bone marrow cells and proliferation index in CTX + LDR mice were higher than those in the untreated mice. LDR or CTX + LDR could induce greater cytochrome c levels and caspase-3 activity in tumors. These results suggest that low dose radiation can enhance the anti-tumor effect of the chemotherapy agent CTX markedly. Furthermore, LDR significantly protects hematopoetic function of the bone marrow, which is of practical significance on adjuvant chemotherapy.     

Cigarette smoking is associated with the reduction of lymphokine-activated killer cell and natural killer cell activities

Lymphokine-activated killer (LAK) cell and natural killer (NK) cell activities were determined in a group of healthy individuals with differing smoking habits. The study population consisted of 54 Japanese males, including 23 smokers, 8 ex-smokers and 23 non-smokers. Peripheral blood mononuclear cells (PBMC) were isolated and used as effector cells. LAK cells were generated by incubation of PBMC with interleukin-2 for 72 h. LAK cell activity against NK-resistant Raji cells and NK cell activity against NK-sensitive K562 cells were examined by 4-h51Cr-release assay. LAK cell activity in the smokers was significantly lower than that in the nonsmokers. The smokers showed significantly lower NK cell activity than the nonsmokers, whereas NK cell activity of the ex-smokers was comparable to that of the non-smokers. The proportion of NK cells (CD3-16+56-, CD3-16-56+, or CD3-16+56+ cells) in the smokers was significantly lower than that in the nonsmokers. The present study demonstrates for the first time that cigarette smokers have lower LAK cell activity than nonsmokers.     

Glutamine requirements in the generation of lymphokine-activated killer cells

The role of glutamine (GLN) in the generation of lymphokine-activated killer (LAK) cell activity was investigated. LAK cells were derived from healthy donors and peripheral blood mononuclear cells (PBMC) were obtained using either unseparated PMBC or DR(-) CD3(-) CD16(+) CD56(+) enriched cells. PBMC were cultured for 6 or 10 days in medium supplemented with recombinant interleukin-2 (rlL-2; 100 U/ml) in the presence of different concentrations of GLN. K562 (natural killer-NK-sensitive targets), 1301 and U-937 (NK-resistant targets) cells were used as targets in the cytotoxic assays. Furthermore, the limiting dilution (LD) culture system was applied as an alternative to the bulk cell culture system. It was found that GLN affects the lytic potential of cultured cells while the frequency of responding cells did not significantly differ between the compared cell cultures performed in the presence of different amounts of GLN. Data on cell proliferation with IL-2 stimulation showed significant differences in cultures performed in the presence or absence of GLN. The results of present investigation suggest a supportive role of GLN in the generation of LAK cells. GLN deficit affects LAK cell killing activity by limiting the number of generated effector cells while acquisition of broad-range killing capacity was not affected.     

IL—2激活的正常人PBL裸鼠体内抗肿瘤作用的研究

给裸鼠尾静脉注射Anip973人肺腺癌细胞造成人工肺转移癌模型。应用IL-2激活的PBL细胞进行体外扩增,诱导出具有杀伤活性的淋巴因子激活的杀伤细胞(LAK细胞),对裸鼠体内抗肿瘤作用进行研究。结果表明注入新鲜淋巴细胞组和单纯注入IL-2组均不能明显抑制肿癌的肺转移,单纯注入LAK细胞组可抑制肿瘤的肺转移,LAK细胞与IL-2同时注射时抑制肺癌转移率比LAK细胞组明显提高。病理检查证实了经LAK细胞和IL-2同时治疗的裸鼠,肺转移癌结节明显减少。LAK细胞与rIL-2伍用亦可显著延长荷瘤裸鼠的生存期。     

rhIL-18对辐照小鼠免疫功能的调节作用

目的研究重组人白细胞介素18(rhIL-18)对辐照小鼠免疫功能的调节作用,探讨rhIL-18在辐照防护中的应用价值。方法将32只小鼠随机分为正常对照组、单纯辐射照射组、rhIL-18+辐射照射组、辐射照射+rhIL-18组,辐照剂量为60Coγ射线4.0Gy,rhIL-18为0.6mg腹腔注射,处理后分别用淋巴细胞转化实验、NK细胞毒实验、T淋巴细胞亚群检测,测定小鼠脾细胞体外培养上清中白细胞介素-2(IL-2)、γ-干扰素(IFN-γ)、单核巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-4(IL-4)和血清中IgG的含量,观察rhIL-18对其免疫功能的调节作用。结果与单纯辐射照射组比较,rhIL-18能提高辐照后小鼠的T、B淋巴细胞转化能力(P0.05),使T、B淋巴细胞转化能力恢复或超过正常对照组水平,辐射照射后注射rhIL-18组的刺激指数达到了2.9〔刀豆蛋白A(ConA)组〕和6.1〔脂多糖(LPS)组〕;可促进辐照所致NK细胞活性抑制的恢复(P0.05),使NK细胞对肿瘤细胞杀伤率达到21.8%～35.6%;上调CD4T细胞数量,达到50个/ml(P0.05),提高辐照小鼠脾细胞分泌IFN-γ、GM-CSF和IL-2的能力(P0.05),但对IL-4分泌能力和IgG产生能力没有调节作用。结论rhIL-18具有促进辐照小鼠免疫功能恢复的作用。