人LAK细胞抗菌杀菌活性多肽HLP—2b的分离纯化

目的：分离纯化人LAK细胞小分子抗菌多肽，并检测其抗肿瘤细胞株K562活性。方法：采用密度梯度离心法分离人外周血单个核细胞，并用IL-2和PHA刺激培养。5%乙酸匀浆细胞获得其酸溶性提取物。应用制备性酸性尿素聚丙烯酰胺凝胶电泳技术和反相高效液相色谱技术分离纯化多肽，Tricine-SDS-PAGE鉴定其分子量，并运用琼脂糖弥散法，MTT法鉴定其抗菌、抗肿瘤活性。结果：从人LAK细胞酸溶性提取物中纯化出一多肽，分子量为7.8kD，命名为HLP-2b，具有抗金黄色葡萄球菌活性和抗肿瘤细胞株K562活性。结论：HLP-2b可能为LAK细胞一新的抗菌抗瘤效应分子。     

人LAK细胞抗菌活性多肽HLP-3P21的分离纯化和鉴定

目的　分离纯化人LAK细胞小分子抗菌多肽 ,鉴定其分子特性 ,检测其抗菌活性。方法　采用密度梯度离心法分离人外周血单个核细胞 ,并用IL 2和PHA刺激培养。 5 %乙酸匀浆细胞获得其酸溶性提取物。应用制备酸性尿素聚丙烯酰胺凝胶电泳技术和反相高效液相色谱技术分离纯化多肽 ,Tricine SDS PAGE鉴定其相对分子质量 (Mr) ,运用琼脂糖弥散法鉴定其抗菌活性 ,采用Edman降解法测定其氮端序列 ,并进行肽质谱分析。结果　从人LAK细胞酸溶性提取物中纯化出一多肽 ,Tricine SDS PAGE表观Mr 为 17× 10 3,命名为HLP 3P2 1(humanlymphocytepeptide) ,具有抗致病性大肠杆菌ML 35P及铜绿假单胞菌ATCC6 5 92 2的活性。其氮端序列为PKRKAEGDAK ,肽质谱的检索分析未发现匹配度大于 5 0 %的蛋白质分子。结论　HLP 3P2 1可能为LAK细胞一新的抗菌效应分子。     

人LAK细胞免疫效应分子HMGN2的鉴定

目的：从人LAK细胞分离纯化和鉴定小分子抗菌多肽。方法：用Hypaque-Ficou梯度离心法从人外周血分离单核白细胞，经IL-2和PHA刺激培养后，5％乙酸匀浆细胞获取酸溶性提取物，应用制备尿素聚丙烯酰胺凝胶电泳技术和反向高效液相色谱技术分离纯化多肽，N-端氨基酸测定和质谱分析鉴定其分子特性。免疫荧光化学、ELISA和Westem Blotting方法进行定位分析。     

人子宫内膜粘液抗菌多肽的分离纯化

目的：分离纯化人子宫内膜粘液抗菌多肽。方法：采用酸性尿素聚丙烯酰胺凝胶电泳琼脂糖弥散法检测子宫内膜粘液酸溶性提取物抗菌活性；琼脂糖电泳洗脱相应抗菌条带，HPLC进一步分离纯化，琼脂糖弥散法检测纯化分子抗菌活性，Tricine-SDS-PAGE电泳检测其分子量。结果：从人子宫内膜酸溶性提取物中纯化出一分子量约为6KD左右的抗菌多肽，对大肠杆菌氨苄青霉素耐药株(E．coli．ML-35P)有抗菌活性。结论：HUP可能是一种新的子宫内膜分泌的抗菌多肽，参与了子宫的天然防御机制。     

人膀胱粘膜抗菌多肽的部分纯化及抗菌活性研究

以５％乙酸冲洗正常人膀胱，获得膀胱粘膜酸溶性提取物。电泳凝胶琼脂糖弥散法抗菌实验分析表明，膀胱粘膜酸溶性提取物中有一条主蛋白带（称之为ＨＢＰ）对致病性大肠杆菌ＭＬ－３５Ｐ株有抗菌活性。以超滤方法对膀胱粘膜酸溶性提取物进行分离纯化，获得分子量低于１×１０~４的超滤纯化样品，其中主要含ＨＢＰ。琼脂糖弥散法抗菌实验结果表明，超滤纯化样品对大肠杆菌ＭＬ－３５Ｐ株、绿脓杆菌ＡＴＣＣ２７８５３株、金黄色葡萄球菌ＡＴＣＣ２５９２３株及血链球菌Ｓ_３４Ｓｒ株均有很强的抗菌活性。采用Ｔｒｉｃｉｎｅ－ＳＤＳ－ｐＡＧＥ鉴定，超滤纯化样品主要显示为两条分子量分别为６．７×１０~３和８．５×１０~３的多肽。研究结果首次提示，人膀胱粘膜内存在抗菌活性很强的内源性抗生肽，是膀胱抗感染防御的主要因素。     

HMGN2分子体外抗乙型肝炎病毒活性研究

目的： 我们先前的研究证明HMGN2（high mobility group nucleosomal-binding domain 2）是人LAK细胞抗菌的一个新的效应分子,本研究欲检测其体外抗乙型肝炎病毒的活性。方法: 应用反向高效液相色谱技术从人淋巴结组织酸溶性提取物中分离纯化批量HMGN2分子,用质谱精确分子量测定、Western blotting和抗菌试验对分离纯化得到的HMGN2分子进行分子鉴定。采用MTT法检测HMGN2分子对稳定转染复制型HBV重组质粒的肝胚瘤细胞株HepG2.2.15细胞的细胞毒性;用不同浓度HMGN2分子作用于HepG2.2.15细胞,分别在第3 d和第6 d收集细胞培养上清液,ELISA检测上清中HBV表面抗原（HBsAg）及e抗原（HBeAg）,采用实时荧光定量PCR法检测上清液HBV DNA的含量。结果: 从人淋巴结组织中分离纯化获得纯度较高的HMGN2蛋白;在检1-100 mg/L范围内,HMGN2分子对HepG2.2.15细胞无细胞毒性;HMGN2分子在1-5 mg/L水平即可显著抑制HBeAg和HBsAg的表达,可显著降低HBV DNA拷贝数。结论: HMGN2分子在体外具有较强的抗HBV活性。     

人LAK细胞抗菌多肽基因重组及其结构与功能的研究

目的和方法 :本研究采用一系列微量肽分离纯化技术及微量抗菌活性检测方法 ,从人LAK细胞中分离纯化获得一个对大肠杆菌和绿脓杆菌具有抗菌作用的活性分子 ,所测得的N -端 10个氨基酸序列为脯氨酸 (Pro ,P)、赖氨酸 (Lys,K)、精氨酸(Arg ,R)、赖氨酸 (Lys ,K)、丙氨酸 (Ala ,A)、天冬氨酸 (Asp ,D)、甘氨酸 (Gly ,G)、谷氨酸 (GluE)、丙氨酸 (Ala ,A)、赖氨酸 (Lys ,K)。应用RACE -PCR技术扩增其cDNA ,获得一个全长编码序列 ,mRNA数据库的检索显示HLP - 3P2 1与HMG - 17分子相同 ,但国内外尚无HMG - 17抗菌功能的报道。为…     

人单核细胞株THP-1中HMGN2的分离纯化

目的:从人单核细胞株THP-1中分离与纯化HMGN2抗菌肽,探讨HMGN2分子抗菌作用。方法:采用HPLC分离纯化,琼脂糖弥散法筛选纯化组分的抗菌活性,对活性组分进行Tricine-SDS-PAGE电泳初步测定分子量。结果:从人THP-1细胞中分离纯化出的HMGN2抗菌肽,对致病性大肠杆菌54080有抗菌活性。结论:HMGN2是一种抗菌分子,可能参与了体内的天然免疫防御作用。     

兔胸腺HMGN2的分离纯化鉴定

目的：分离纯化鉴定兔高迁移率非组蛋白N2（High mobility group chromosomal proteinN2，HMGN2）。方法：利用液氮研磨和5％高氯酸从新鲜兔胸腺组织获得酸溶性萃取物，经反相高效液相色谱（RP-HPLC）分离得到HMGN2，经过SDS-聚丙烯酰胺凝胶电泳和Western-blotting对HMGN2进行分子量测定和特异性鉴定。并利用琼脂糖弥散抑菌实验对其杀菌活性进行鉴定。结果：利用上述方法分离纯化出高纯度的兔HMGN2。对大肠埃希菌临床分离株54080、大肠埃希菌ATCC25922有明显杀菌活性。结论：建立简单分离高纯度的HMGN2的方法。     

家蝇源抗K562活性物质的分离与纯化

为从家蝇Ⅲ龄幼虫体内提取具有抗K562细胞活性的物质,并对其组分化学性质及对人正常细胞的作用进行分析。本文用大肠杆菌诱导家蝇Ⅲ龄幼虫大量表达活性物质,然后通过研磨、离心、固相萃取法（SPE）初步分离活性物质,通过反相高效液相色谱（RP—HPLC）进一步纯化提取活性组分,通过四甲基偶氮噻唑蓝（Methyl thiazolyl tetrazolium,MTT）比色法和光镜观察法相结合,筛选出对K562有抑制作用的活性组分,并鉴定其对人正常细胞293T的作用。结果显示,从家蝇Ⅲ龄幼虫体内分离出4个具有抑制K562细胞活性的多肽组分（峰5～8）,具有分离度高、纯度高等特点。当质量浓度为100ug／mL和作用时间为24h时,4个多肽组分均具有抗K562细胞的活性,其中峰5多肽组分和峰8多肽组分活性较强,对K562细胞的相对抑制率分别为48．52％和65．80％。结果表明,家蝇Ⅲ龄幼虫体内存在一些具有抗K562细胞活性的多肽,而且对人正常细胞几乎无杀伤作用。但是,这些多肽是否属于抗肿瘤肽还有待被证实。     

人单核细胞系THP-1中HMGN2的分离纯化及其抗菌活性

 目的 从人单核细胞株THP-1中分离与纯化HMGN2抗菌肽,应用微量琼脂糖弥散法检测抗菌活性,以探讨HMGN2分子的抗菌作用。方法 采用HPLC分离纯化, 琼脂糖弥散法筛选纯化有抗菌活性的组分,用Tricine-SDS-PAGE电泳初步测定其分子质量,以质谱法进行分子质量的精确分析。结果 从人THP-1细胞中分离纯化出HMGN2抗菌肽, 琼脂糖弥散法检测显示其对大肠杆菌氨苄青霉素耐药株ML-35p有较强的抑菌活性,对大肠杆菌标准株ATCC25922和临床分离株54080亦有较强的抗菌活性,而对金黄色葡萄球菌ATCC25923未检测出抗菌活性。结论：HMGN2是一种抗菌分子,可能参与了体内的天然免疫防御作用。     

五谷虫抗金黄色葡萄球菌物质的纯化及抗菌机制

背景：五谷虫抗菌物质提纯方法存在争议,抗菌作用机制不清。 目的：分离纯化五谷虫抗菌物质,并明确其抗菌机制。 方法：应用超滤法对五谷虫粗提物进行纯化,用酶标浊度法及K-B纸片琼脂扩散法筛选出具有明显抗菌活性的滤出液;MTT法检测滤出液对金黄色葡萄球菌增殖的影响及测定最低抑菌浓度(MIC);应用电镜技术观察抗菌物质作用后金黄色葡萄球菌的形态变化。 结果与结论：酶标浊度法及K-B纸片琼脂扩散法均显示与五谷虫粗提物及截留液相比较,滤出液   (Mr<10 000)的抗菌活性最强(P < 0.05),能明显抑制金葡萄菌增殖,且对金黄色葡萄球菌的最低抑菌浓度(MIC)为1 g/L。五谷虫滤出液抗菌物质能使金黄色葡萄球菌细胞膜的通透性增加,使细菌细胞膜破裂,细胞内容物渗出。五谷虫抗菌物质相对分子质量小于10 000,其可能通过作用于金黄色葡萄球菌细胞膜而形成孔洞,增加细胞膜通透性,而发挥抗菌作用。     

Gelatin sponge model of effector recruitment: tumoricidal activity of adherent and non-adherent lymphokine-activated killer cells after culture in interleukin-2

This study examined the specific tumoricidal activity of lymphokine-activated killer (LAK) cells derived from tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors. A pre-implanted gelatin sponge was employed to capture infiltrating host effectors during the expression of concomitant tumor immunity. Additionally, this study compared the cytolytic activity of these sponge-derived cells with those of counterpart splenic lymphocytes. The cells from both sources were cultured for 4 days in IL-2 to generate LAK cells which were further expanded in IL-2-containing medium for up to 11 days. The cytotoxic activities of these cells were measured in a Chromium-51 release assay. The data revealed that the culture of splenic, or sponge-derived lymphocytes results in the emergence of non-adherent and adherent cell populations with LAK activity. The 4-day sponge-derived LAK cells (adherent and non-adherent) exhibited significant cytolysis of EMT6 cells while the spleen-derived counterparts showed minimal cytotoxicity toward these targets. Some NK activity in LAK cells derived from both sources was evident by their lysis of YAC-1 cells. LAK cells from both sources were incapable of lysing histo-compatible EL-4 (H-2b) tumor cells. The lysis of the EMT6 cells by the sponge-derived LAK cells was maintained over an 11-day period of culture in IL-2. Conversely, the spleen-derived LAK cells were unable to significantly lyse EMT6 cells during this period of in vitro culture. These results show the superior specific tumoricidal activity of LAK cells derived from lymphocytes mediating tumor rejection in vivo (sponge-derived) over that of counterpart splenic lymphocytes.     

The importance of splenectomy for the adoptive immunotherapy of cancer

Adoptive immunotherapy (AIT) of cancer using lymphocytes cultured in vitro in interleukin-2 (LAK cells) has gained much attention due to the high level of broad anti-tumor activity both in vitro and in vivo. A major hypothesis has suggested that if unfractionated lymphoid cells grown in IL-2 can mediate significant anti-tumor effects then purified LAK effector cells should yield even higher activity. Furthermore, devising new techniques to achieve optimal expansion of the purified LAK effector cells would further improve this form of immunotherapy. In this report we present data on a new method for purifying and expanding LAK effector cells obtained from the peripheral blood and spleen. The results indicate that as a source of LAK cells, the spleen is superior both quantitatively ani qualitatively when compared to peripheral blood and should be seriously considered as the source of cells for AIT of cancer.     

Capacity of tumor necrosis factor to augment lymphocyte-mediated tumor cell lysis of malignant mesothelioma

Recombinant human tumor necrosis factor (rHuTNF) was evaluated both for direct anti-tumor action against human malignant mesothelioma and for its capacity to augment the generation and lytic phases of lymphocyte-mediated cytotoxicity against this tumor. rHuTNF was directly toxic by MTT assay to one of two mesothelioma cell lines evaluated, but had no effect on susceptibility to subsequent lymphocyte-mediated lysis of either line. TNF alone was incapable of generating anti-mesothelioma lymphokine-activated killer cell (LAK) activity. Furthermore, it did not augment the degree or LAK activity produced by submaximal interleukin-2 (IL-2) concentrations nor did it augment lysis of mesothelioma cells by natural killer (NK) or LAK effector cells during the 4-hr 51chromium release cytolytic reaction. The studies also suggest that mesothelioma targets are less responsive to TNF plus submaximal IL-2 concentrations than the standard LAK sensitive target Daudi, raising the possibility that intermediate LAK sensitive tumors such as mesothelioma may require separate and specific evaluation in immunomodulation studies. This in vitro study indicates that use of low-dose rHuTNF and IL-2 is unlikely to be an effective substitute for high-dose IL-2 in generation and maintenance of LAK activity in adoptive immunotherapy for mesothelioma.     

抗NKG2D多克隆抗体抑制NK和LAK细胞细胞毒效应的研究

目的 :分析抗NKG2D多克隆抗体 ( pAb)对NK和LAK细胞毒作用的影响。方法 :应用密度梯度离心法分离外周血单个核细胞 (PBMC) ,经 10mg/LPHA和 1× 10 6U/LrhIL 2诱导LAK细胞产生 ,再应用流式细胞术 (FCM)分选NK细胞并进行表型检测。加入抗NKG2DpAb封闭NK和LAK细胞表面的NKG2D分子后 ,用MTT比色法检测其细胞毒效应。结果 :经FCM分析证实 ,获得高纯度、高活性的NK细胞。抗NKG2DpAb能显著抑制NK和LAK细胞对K5 6 2、HepG2细胞的细胞毒效应。NK细胞对两种靶细胞的细胞毒效应分别下降了 82 .9%和 75 .6 % ;LAK细胞对两种靶细胞的细胞毒效应分别下降了 5 2 .8%和 5 0 .2 %。但抗NKG2DpAb不能显著抑制两种效应细胞对人鼻咽癌细胞系CNE的细胞毒效应。结论 :抗NKG2DpAb可通过封闭NK和LAK细胞表面的NKG2D分子 ,抑制其对肿瘤细胞的细胞毒效应     

IL-2和IL-15协同调节T细胞的增殖和LAK细胞的杀伤活性

目的 探讨IL-2和IL-15在免疫调控和应答中的协同作用。方法 IL-2、IL-15和IL-2 IL-l5组刺激CTLL细胞的增殖，^3H—TdR掺入法测cpm值；IL-2、IL-15和IL-2 IL-l5组诱导PBMC中NK和LAK细胞的发育，4h^51Cr释放实验检测对K562和LiBr细胞的杀伤活性。结果 IL-2和IL-15都能诱导CTLL认细胞的增殖和NK、LAK细胞的杀伤活性，IL-2和IL-15可明显协同上调CTLL认的增殖和LAK细胞的杀伤活性。结论 IL-2和IL-15在免疫调控和应答中有一定的协同作用，为细胞因子联合应用于临床治疗肿瘤等疾病提供了实验依据。