抗肝癌hdsFv融合RC-RNase重组免疫毒素的表达、纯化及活性测定

目的制备有临床应用前景的特异性高、稳定性强的新型抗肝癌重组免疫毒素。方法利用IPTG诱导抗肝癌hdsFv-RC-RNase重组免疫毒素在大肠杆菌中表达。表达产物经Ni-NTA亲合层析法纯化并复性,应用免疫细胞化学和MTT的方法分别检测其对人肝癌细胞的特异性结合和杀伤活性。结果重组免疫毒素以可溶性形式在大肠杆菌中获得表达。免疫细胞化学和细胞毒实验表明其能够特异性地结合并杀伤肝癌细胞,并且能够保持较强的稳定性。结论我们抗肝癌hdsFv-RC-RNase重组免疫毒素的成功制备,为其进一步的临床应用研究奠定了一定的实验基础。     

两种基因重组免疫毒素导向治疗乳腺癌的细胞毒活性研究

目的:探讨HELβ1-PE33、HELβ1-PE38KDEL两种基因重组免疫毒素对乳腺癌细胞的细胞毒活性。方法:构建亚克隆质粒pHELβ1-PE33;诱导生成、纯化HELβ1-PE33蛋白,并用凝胶电泳法检测其与HELβ1-PE38KDEL蛋白纯度;采用MTT法、软琼脂集落形成法检测这两种蛋白对乳腺癌细胞增殖的影响,比较两种蛋白在不同的乳腺癌细胞系中的细胞毒活性。结果:纯化后的两种蛋白均有导向作用,其纯度均超过98%。在免疫毒素敏感乳腺癌细胞内,MTT实验表明HELβ1-PE38KDEL对DY36T2、MDA-MB-453、47T1LZ1、BT474M1、SKBR3的IC50分别为(0.25±0.02)ng/ml、(0.35±0.05)ng/ml、(0.02±0.02)ng/ml、(0.02±0.02)ng/ml、(20.3±0.01)ng/ml,而HELβ1-PE33的IC50分别为(5.90±0.05)ng/ml、(3.91±0.11)ng/ml、(0.09±0.02)ng/ml、(0.06±0.05)ng/ml、(125.00±0.12)ng/ml、(10.80±0.01)ng/ml,两者的细胞毒活性在统计学上有显著意义(P<0.01);软琼脂集落形成实验表明HELβ1-PE38KDEL对MDA-MB-453、BT474M1的IC50分别为(0.05±0.01)、(0.005±0.000)ng/ml,而HELβ1-PE33对上述两种细胞的IC50则为(5.00±0.02)ng/ml和(0.05±0.01)ng/ml,二者的细胞毒活性在统计学上有显著意义(P<0.01)。HELβ1-PE33和HELβ1-PE38KDEL对乳癌细胞抑制作用的时间效     

抗肝癌重组免疫毒素对荷人肝癌裸鼠移植瘤生长的抑制作用

目的:观察二硫键稳定人源化抗肝癌单链抗体(humanized disulfide stabilization single chain antibody,hdsFv)融合牛蛙核糖核酸酶(rana catesbeiana ribonuclease,RC-RNase)重组免疫毒素(hdsFv-RC-RN-ase)对荷人肝癌裸鼠移植瘤生长的抑制作用。方法:将人肝癌细胞系SMMC-7721细胞接种于裸鼠皮下,建立荷人肝癌裸鼠移植瘤动物模型,随机分为3组,分别经尾静脉注射给予生理盐水、盐酸多柔比星和抗肝癌hdsFv-RC-RNase治疗,疗程为2周。通过测量各实验组裸鼠肿瘤体积及瘤质量变化,绘制肿瘤生长曲线并计算抑瘤率。治疗结束后取各组裸鼠肿瘤组织及重要器官HE染色,光学显微镜下观察。结果:治疗后hdsFv-RC-RNase组与空白对照组相比较,荷人肝癌裸鼠移植瘤生长速度显著减慢,肿瘤体积和瘤质量明显减小,P<0·01;同样,与盐酸多柔比星组相比较差异有统计学意义,P<0·01。hdsFv-RC-RNase组和盐酸多柔比星组抑瘤率分别为(78·9±4·1)%和(70·3±6·6)%,P<0·01。光学显微镜下观察hdsFv-RC-RNase组和盐酸多柔比星组肿瘤组织出现明显坏死,尤以前者更为显著。各实验组裸鼠重要器官未见明显异常。结论:抗肝癌hdsFv-RC-RNase重组免疫毒素对荷人肝癌裸鼠移植瘤生长具有良好的抑制作用。     

免疫毒素的研究进展

免疫毒素用于恶性肿瘤的导向治疗经经取得了明确的疗效。本文简要综述近年来免疫毒素的一些研究进展。     

免疫毒素的研究进展

免疫毒素用于恶性肿瘤的导向治疗已经取得了明确的疗效.本文简要综述近年来免疫毒素的一些研究进展.     

免疫毒素的研究进展

肿瘤的导向治疗开创了肿瘤治疗的新篇章.在众多的导向治疗中免疫毒素发展较为迅速,尤其是利用现代生物技术,分别将起导向作用的载体和杀伤效应的毒素基因进行一系列的改建,然后重组而获得的免疫毒素有了长足的发展,许多免疫毒素已经进入临床Ⅰ,Ⅱ期试验,显示出良好的应用前景.     

重组免疫毒素治疗实体瘤的研究进展

0引言 免疫毒素(Immunotoxins,ITs),是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)偶联而成的具有特异性细胞杀伤能力的杂合分子[1].     

抗肿瘤基因工程单链抗体及其免疫毒素的研究

抗体VH基因和VL基因经寡核苷酸接头连接成ScFv基因，转入原核或真核表达系统中表达ScFv。ScFv具有亲本抗体相同的抗原结合特性和能力．且抗原性大大减弱。ScFv分子量小,易穿入肿瘤和实体组织内部，也易进入病毒峡谷。ScFv与药物、毒素、同位素或酶等偶联构成的导向治疗制剂在血中清除迅速，不残留于正常组织，尤其不在肾脏中积聚，因而毒副作用很小。PCR技术的应用，使制备单链免疫毒素在二周内即可实现，大大缩短了导向药物的制备时间。改善培养条件，E-coli能表达并装配和折叠成具有生物活性的双价ScFv及其免疫毒素。     

晚期原发性肝细胞癌的药物治疗

原发性肝癌（PLC）是临床上常见的消化系统恶性肿瘤之一,90%以上为肝细胞癌（HCC）。大多数原发性肝细胞癌患者存在肝炎基础疾病,肝功能差,局部治疗后易复发,确诊时已到晚期,进展快,预后极差。晚期原发性肝细胞癌系统治疗包括基础肝病治疗、系统化疗、分子靶向治疗、免疫治疗,本文就晚期原发性肝细胞癌的系统化疗、分子靶向治疗、免疫治疗作一综述。     

A bivalent recombinant immunotoxin with high potency against tumors with EGFR and EGFRvIII expression

EGFR and EGFRvIII are overexpressed in various types of cancer, serving as optimal targets for cancer therapy. Capitalizing on the high specificity of humanized antibody 806 (mAb806) to the EGFR and EGFRvIII overexpressed in cancer, we designed and generated a bivalent recombinant immunotoxin (RIT, DT390-BiscFv806) by fusing the mAb806-derived bivalent single-chain variable fragment with a diphtheria toxin fragment, DT390. In vitro, DT390-BiscFv806 efficiently internalized into the cells and exhibited high cytotoxicity against the U87 glioblastoma cells and the EGFRvIII-transfected U87 (U87-EGFRvIII) cells with a half maximal inhibition concentration (IC₅₀) of 1.47 nM and 2.26 × 10⁻⁴ nM, respectively. Notably, DT390-BiscFv806 was 4 orders of magnitude more potent against the U87-EGFRvIII cells than against the parent U87 cells. The cytotoxicity against a group of 6 head and neck squamous cell carcinoma cell lines were further analyzed, showing an IC₅₀ ranging from 0.24 nM to 156 nM, depending on the expression level of EGFR/EGFRvIII. In animals, the U87-EGFRvIII tumor xenografts grew extremely faster than the parental U87, and systemic administration of DT390-BiscFv806 significantly inhibited the growth of established U87-EGFRvIII and U87 tumor xenografts, showing a growth inhibition rate of 76.3% (59.82–96.2%) and 59.4% (31.5–76.0%), respectively. In pathology, the RIT-treated tumors exhibited a low mitotic activity and a large number of degenerative tumor cells, compared with the control tumors. The results indicate that DT390-BiscFv806 is promising for treatment of various types of cancer, especially for those with high EGFR expression or with EGFR and EGFRvIII co-expression.     

Radiation therapy for bone metastasis of hepatocellular carcinoma

Background The efficacy of radiation therapy for bone metastasis of hepatocellular carcinoma was examined retrospectively. Methods The effect of radiation therapy for pain and paresis was evaluated in 38 patients who received planned radiation therapy with the total dose ranging from 26 to 60 Gy. The percentage regression of 14 metastatic bone lesions was calculated by using computed tomography: percentage regression=[(pre-radiation therapy tumor volume—postradiation therapy tumor volume)/pre-radiation therapy tumor volume]×100. Results The 1-, 2-, and 5-year survival rates of the 38 patients from the start of the radiation therapy were 30% 18%, and 9.0% respectively. The median survival period was 172 days. Pain relief was obtained in 91% of 44 painful bone metastases. No correlation was observed between the percentage regression and the tumor size. Complete regression of bone metastasis was noted in 6 of 14 patients who could be evaluated with computed tomography. Three of the 6 paiients with complete response have survived for more than 6 years after radiation therapy. Conclusion Radiation therapy is an effective method for palliation of bone metastasis. Although the prognosis of most patients with bone metastasis from hepatocellular carcinoma is poor, some attain longterm survival after treatment.     

肝细胞癌的诱导分化治疗

肝细胞癌是最常见的恶性肿瘤之一,虽然其诊断和治疗有不少进展,但其预后仍较差,病死率较高。诱导分化治疗这一概念的提出为肝细胞癌的治疗指明了新的方向。诱导分化治疗在血液系统肿瘤治疗方面获得了成功,其经典范例是全反式维甲酸临床治疗急性早幼粒白血病;但是其在恶性实体肿瘤领域的进展远不如血液系统肿瘤。目前特异性的肝细胞癌诱导分化剂较少,相关的临床应用更是有限。靶向性地针对肿瘤干细胞分化相关的信号转导通路或转录因子进行干预可能起到诱导肝细胞癌分化的效果,例如通过上调与肝细胞分化密切相关的转录因子肝细胞核因子4α（hepatocyte nuclear factor 4α, HNF4α)来诱导肝癌细胞,特别是诱导肝癌干细胞向成熟阶段分化,已初见成效。肿瘤发生发展过程中存在诸多表观遗传学异常改变,如甲基化、乙酰化水平异常或microRNA表达异常,有些改变与肿瘤分化密切相关,干预这些异常改变的药物如组蛋白去乙酰化酶抑制剂对肿瘤起到一定的诱导分化作用。因此,肿瘤干细胞学说的出现及表现遗传学的发展给肝细胞癌诱导分化治疗研究提供了具体的途径。     

Proton beam therapy for hepatocellular carcinoma

Introduction: Radiation therapy is an effective treatment option for hepatocellular carcinoma (HCC) patients. However, radiotherapy for HCC still has limited recognition as a standard treatment option in international consensus guidelines due to a paucity of randomized controlled trials and the risk of hepatotoxicity, which is primarily mediated by baseline liver function and dose delivered to non-tumor liver cells. Proton beam therapy (PBT) may offer advantages over photon-based radiation treatments through its dosimetric characteristic of sparing more liver volume at low to moderate doses. PBT has the potential to reduce radiation-related hepatotoxicity and allow for tumor dose escalation.

Areas covered: This article reviews the clinical rationale for using PBT for HCC patients and clinical outcome and toxicity data from retrospective and prospective studies. PBT-specific technical challenges for these tumors and appropriate selection of patients to be treated with PBT are discussed.

Expert commentary: Local control, overall survival, and toxicity results are promising for liver PBT. Future studies, including ongoing randomized cooperative group trials, will aim to determine the incremental benefit of PBT over photons and which patients are most suitable for PBT.     

AFP启动子与胸苷激酶基因治疗肝癌的实验研究

目的：探讨AFP启动子与胸苷激酶疗法对肝癌的作用。方法：采用含tk基因与潮霉素磷酸转移酶hy基因融合基因的真核表达载体及仅含潮霉素磷酸转移酶hy的空载体,以脂质体为介导,将这两种质粒分别转染肝癌细胞系HEPG2。潮霉素B药物敏感实验筛选HEPG2,不同浓度丙氧鸟苷分别作用于HEPG2,HEPG2-AFP—hytk及HEPG2-hy。结果：潮霉素B药物敏感实验筛选HEPG2,12天时HEPG2全部死亡的最低浓度为60μg／ml,30μmol／L丙氧鸟苷可最大限度杀死HEPG2-AFP—hytk。杀伤效应随时间而增强,作用24小时后开始出现毒性作用,至96小时死亡率〉80％。仅含有18％的HEPG2-AFP—hytk就可使周围20％的HEPG2细胞死亡,总死亡率为38％。结论：60μg／ml潮霉素是筛选HEPG2－AFP—hytk及HEPG2－hy阳性克隆的最佳剂量。30μmol／L丙氧鸟苷是作用于HEPG2－AFP—hytk的最佳剂量。HSV—tk／GCV系统大于18％HEPG2-AFP—hytk开始具有旁观者效应,可提高疗效。     

以外科手术为主综合治疗小肝癌134例临床报告

背景与目的：手术切除是治疗小肝癌的首选方法,但术后5年复发率高达35．4％－45．3％,是影响手术疗效的关键因素。本研究拟探讨以外科为主的综合治疗来降低小肝癌术后复发率。方法：以外科为主综合治疗小肝癌（直径≤5cm）患者134例,男119例,女15例,年龄18－70岁,中位年龄45岁;手术切除（切除组）121例,其中,不规则性肝叶切除16例,局部切除83例,肝叶或肝段切除12例,左半肝切除2例,联合胆吓切除8例;切除后切缘注射无水酒精或用渗入无水酒精的明胶海绵包埋于瘤床共22例。手术不能切除13例,行肝固有动脉结扎合并肝动脉及门静脉双插管化疗或瘤体内注射无水酒精或冷冻、射频治疗、微波固化、栓塞化疗等。结果：小肝癌手术切除率90．3％,无手术死亡。切除组术后1、3、5、10年生存率分别为89．3％、74．4％、64．6％和43．8％;术后1、3、5年复发率分别为11．9％、23．8％和32．1％。全组1、3、5、10年生存率分别为88．8％、72．2％、63．4％和41．7％;1、3、5年复发率分别为15．9％、29．1％和36．6％。结论：手术切除是治疗小肝癌的有效方法,以个体化为原则外科为主的综合治疗可降低术后复发率,提高小肝癌治疗效果。     

肝细胞肝癌肾上腺转移的放射治疗

目的探讨肝癌肾上腺转移的放射治疗效果.方法回顾性总结本院22例肝癌肾上腺转移病人,用6MX或15MV光子,进行肾上腺病灶外照射,剂量36～54Gy,中位剂量50Gy.结果14例肾上腺转移病人出现疼痛症状,外放疗后11例(78.6%)疼痛得到完全缓解;2例(14.3%)得到明显缓解,但仍需要止痛药治疗;73%(15/22)病例的肿瘤明显缩小;全部病例中位生存期为10个月;未出现因肾上腺转移灶致死病例;副作用轻微.结论肝细胞肝癌肾上腺转移对放射治疗敏感,50Gy的放疗剂量是比较安全的姑息性治疗.     

Latest developments in targeted therapy for hepatocellular carcinoma

The advent of sorafenib can be considered as a turning point in the history of advanced hepatocellular carcinoma. After unfortunate attempts at using chemotherapy, drugs targeting key pathways have generated new perspectives in this field. This means not only killing both tumor cells and cirrhotic fragile tissue, but killing them selectively; more than was previously possible. This seems like the Copernican Revolution. However, hepatocellular carcinoma is pathogenetically complicated, resulting from the number of mutations. Until now, there has not been a single drug able to block and reverse abnormally activated signaling in hepatocellular carcinoma cells. In this article, we describe the most promising targeted drugs being studied in hepatocellular carcinoma and depict the possible future scenarios.