卵巢癌单克隆抗体—脂质体—药物交联物制备及其体…

以我室自制卵巢上皮癌单克隆抗体ＣＯＣ１６６－９与包载阿霉素或顺铂的脂质体结合，制备出单克隆抗体（单抗）脂质体阿霉素交联物（ＭＬＡ）和单抗脂质体顺铂交联物（ＭＬＰ）。并用ＭＬＡ和ＭＬＰ对卵巢癌细胞系ＳＫＯＶ３进行生长抑制实验，结果表明，在较高浓度时ＭＬＡ与阿霉素的杀伤ＳＫＯＶ３细胞能力相同；当浓度减低时，ＭＬＡ明显强于阿霉素。而ＳＫＯＶ３对ＭＬＰ和顺铂都不敏感。     

卵巢癌可溶性抗原和抗CD3单克隆抗体诱导细胞毒性T细胞抗人卵巢癌的实验研究

目的探讨卵巢癌过继细胞免疫治疗新方法。方法提取卵巢癌细胞（ＣＯＣ１和ＣＯＣ２ｎ）可溶性抗原（ＴＳＡ），用ＴＳＡ和抗ＣＤ３单克隆抗体（ＣＤ３ＭｃＡｂ）共同诱导正常人外周血单个核细胞（ＰＢＭＣ）产生细胞毒性Ｔ细胞（ＣＴＬ），用ＣＴＬ于体外杀伤ＣＯＣ１细胞和裸鼠体内抑制ＣＯＣ２ｎ移植瘤的生长，体外和淋巴因子激活杀伤细胞（ＬＡＫ）、淋巴因子和抗ＣＤ３单抗激活的杀伤细胞（ＣＤ３ＡＫ）细胞进行比较，体内和ＣＤ３ＡＫ细胞进行比较。结果ＣＴＬ、ＣＤ３ＡＫ和ＬＡＫ对ＣＯＣ１细胞的细胞毒作用分别为７９．４％、５２．１％和５１．７％（Ｐ＜０．０１）。在体内，ＣＴＬ与ＣＤ３ＡＫ和未经处理的对照组比较，卵巢癌细胞移植到裸鼠体内的第９天，肿瘤平均体积分别为４４．４±２４．２ｍｍ３、１１８．８±４０．０ｍｍ３和４４３．０±１５８．７ｍｍ３（Ｐ＜０．０１），裸鼠平均生存期分别为２８．５天、２５．５天和１７天（Ｐ＜０．０１）。结论本方法诱导产生的ＣＴＬ，在体内、体外对卵巢癌细胞均有较高的细胞毒作用，能明显抑制肿瘤生长，为卵巢癌治疗提供了新的思路。     

脂质体—C—erbB2反义脱氧寡核苷酸对人卵癌裸鼠网膜移植瘤 …

目的 探讨脂质体－Ｃ－ｅｒｂＢ２反义脱氧寡苷酸（Ｃ－ｅｒｂＢ２ Ｓ－ＯＤＮｓ２）对人卵巢癌裸鼠网膜移植瘤生长及顺铂（ＤＤＰ）敏感性的影响。方法 对２０只裸鼠建立人卵巢癌裸鼠网膜移植模型,分为对照组（腹腔注射无菌水）、观察组（腹腔注射脂质体－Ｃ－ｅｒｂＢ２ Ｓ－ＯＤＮｓ）、化疗组（腹腔注射无菌水及ＤＤＰ）、观察＋化疗组（腹腔注射脂质体－Ｃ－ｅｒｂＢ２ Ｓ－ＯＤＮｓ及ＤＤＰ）。治疗期间观测裸鼠体重变化     

单纯疱疹病毒胞腺嘧啶核苷激酶基因羟甲基无环岛苷系对人卵 …

观察单纯疱疹病毒胸腺嘧啶核苷激素酶基因羟甲基无环鸟苷系统对人卵巢癌的体内治疗作用。方法先将人卵巢癌细胞ＡＯ及携有ＨＳＶ１－ｔｋ基因的ＡＯ注射于裸鼠皮下,形成肿瘤后再移植于裸鼠网膜,并以病毒生物细胞的培养液对ＡＯ网膜移植瘤进行体风转染,然后以ＧＣＶ对体外ＡＯ／ＨＳＶ１－ｔｋｃ转移的肿瘤及经体内转染的ＡＯ肿瘤进行治疗。     

单纯疱疹病毒胸腺嘧啶核苷激酶基因/羟甲基无环鸟苷系统对人卵巢癌裸鼠网膜移植瘤的作用

目的观察单纯疱疹病毒胸腺嘧啶核苷激酶基因（ＨＳＶ１ｔｋ）／羟甲基无环鸟苷（ＧＣＶ）系统对人卵巢癌的体内治疗作用。方法先将人卵巢癌细胞ＡＯ及携有ＨＳＶ１ｔｋ基因的ＡＯ细胞（ＡＯ／ＨＳＶ１ｔｋｃ）注射于裸鼠皮下,形成肿瘤后再移植于裸鼠网膜,并以病毒生产细胞（ＶＰＣ／ＨＳＶ１ｔｋｃ）的培养液对ＡＯ网膜移植瘤进行体内转染,然后以ＧＣＶ对体外ＡＯ／ＨＳＶ１ｔｋｃ转移的肿瘤及经体内转染的ＡＯ肿瘤进行治疗。结果ＨＳＶ１ｔｋ／ＧＣＶ基因治疗系统对ＡＯ裸鼠网膜移植瘤的抑制率为４６．８％,而ＡＯ／ＨＳＶ１ｔｋｃ裸鼠网膜移植瘤经ＧＣＶ治疗后,多数仅残留显微镜下可见的癌灶。结论ＧＣＶ在腹腔内对携有ＨＳＶ１ｔｋ基因的肿瘤具有更为显著的杀伤效应;ＨＳＶ１ｔｋ／ＧＣＶ基因治疗系统在卵巢癌的治疗上可能具有良好的应用前景。     

卵巢癌可溶性抗原和抗CD3单克隆抗体诱导细胞毒性T细胞抗人 …

探讨卵巢癌过继细胞免疫治疗瓣方法。方法 提取卵巢癌细胞可溶性抗原,用ＴＳＡ和抗ＣＤ３单克隆抗体共同诱导正常人外周血单个核细胞产生细胞毒性Ｔ细胞,用ＣＴＬ于体外杀伤ＣＯＣ１细胞和裸鼠体内抑制ＣＯＣ２ｎ移植瘤的生长,体外和淋巴因子激活杀伤细胞,淋巴因子和抗ＣＤ３单抗激活的杀伤细胞细胞进行比较,体内和ＣＤ３－ＡＫ细胞进行比较。     

人卵巢癌裸鼠腹腔及网膜移植的研究

目的：建立既符合卵巢癌临床特征，又便于实验观察和分析的人卵巢癌裸鼠异种移植模型。方法：将体外培养的卵巢癌Ａｏ细胞接种于裸鼠皮下和腹腔内，并切取皮下移植瘤；在麻醉状态下对裸鼠进行网膜局部手术移植。结果：腹腔种植成瘤率为７０％（７／１０），肿瘤结节大小不一，分布广泛，其中２只腹水形成；网膜移植全部成瘤，移植瘤呈单一生长，易分离和切除，肿瘤重量一致性较好。结论：人卵巢癌裸鼠网膜异种移植模型直接反映肿瘤生物学行为，便于观察和分析实验结果，适于在卵巢癌实验研究中应用。     

必解癌Ⅱ号在荷人卵巢癌裸鼠体内的抗肿瘤作用

用我所研制的必解癌Ⅱ号（ＢＪＡ－Ⅱ）在高转移人卵巢癌裸鼠皮下移植瘤模型上进行抗肿瘤治疗（ＢＪＡ－Ⅱ组），并与顺铂治疗（顺铂组）、ＢＪＡ－Ⅱ和顺铂治疗（联合用药组）及空白对照组（对照组）比较。治疗于裸鼠接种移植瘤的次日开始，将ＢＪＡ－Ⅱ按每天每只裸鼠１６ｍｇ的剂量混入饲料内经口投入，治疗５３天，每只裸鼠服药总量达８４８ｍｇ。结果：ＢＪＡ－Ⅱ抑制肿瘤生长，抑瘤率６３．７％，有２只裸鼠移植瘤消失。实验结束，ＢＪＡ－Ⅱ组平均瘤重与对照组比较，差异有显著意义（Ｐ＜０．０５）；ＢＪＡ－Ⅱ有抗肿瘤转移作用（ＢＪＡ－Ⅱ组中１只转移，对照组中４只转移）。ＢＪＡ－Ⅱ组治疗后周围血白细胞数量、自然杀伤细胞活性、脾重／体重比值、淋巴结窦性组织细胞增生等指标均优于顺铂组。表明：ＢＪＡ－Ⅱ在裸鼠体内具有抑制肿瘤生长和抗肿瘤转移、提高宿主免疫功能的作用。     

脂质体-C-erbB2反义脱氧寡核苷酸对人卵巢癌裸鼠网膜移植瘤生长及化疗药物敏感性的影响

目的探讨脂质体ＣｅｒｂＢ２反义脱氧寡核苷酸（ＣｅｒｂＢ２ＳＯＤＮｓ）对人卵巢癌裸鼠网膜移植瘤生长及顺铂（ＤＤＰ）敏感性的影响。方法对２０只裸鼠建立人卵巢癌裸鼠网膜移植瘤模型,分为对照组（腹腔注射无菌水）、观察组（腹腔注射脂质体ＣｅｒｂＢ２ＳＯＤＮｓ）、化疗组（腹腔注射无菌水及ＤＤＰ）、观察＋化疗组（腹腔注射脂质体ＣｅｒｂＢ２ＳＯＤＮｓ及ＤＤＰ）。治疗期间观测裸鼠体重变化,治疗结束后取瘤体称重进行电镜观察。结果观察组抑瘤率为３７％,电镜下可见细胞异染色质增多,线粒体退化。观察＋化疗组抑瘤率可达５０％。观察组裸鼠体重无明显改变。结论ＣｅｒｂＢ２反义基因治疗在卵巢癌基因治疗中有一定作用,与化疗联合应用可以取得更好的治疗效果。     

脂质体-C-erbB_2反义寡核苷酸对人卵巢上皮癌裸鼠皮下移植瘤的作用

目的：探讨经脂质体—硫代磷酸化修饰的Ｃ－ｅｒｂＢ２反义脱氧寡核苷酸（Ｃ－ｅｒｂＢ２ｓｕｌｆｉｄｅ－ＯＤＮｓ）作用后的卵巢上皮癌细胞在裸鼠皮下的成瘤能力及形态学变化。方法：利用离体途径将Ｃ－ｅｒｂＢ２ｓｕｌｆｉｄｅ－ＯＤＮｓ导入卵巢上皮癌细胞，然后接种于裸鼠皮下，观察肿瘤体积的变化，并计算抑瘤率。用透射电镜观察肿瘤形态变化。结果：经脂质体－Ｃ－ｅｒｂＢ２ｓｕｌｆｉｄｅ－ＯＤＮｓ作用后的卵巢上皮癌细胞在裸鼠皮下成瘤能力降低，最大抑瘤率可达４１．７％。超微结构显示实验组肿瘤细胞异染色质增多，分化增高。结论：Ｃ－ｅｒｂＢ２癌基因的反义调控能降低卵巢上皮癌的成瘤能力，抑制肿瘤生长，在卵巢上皮癌的基因治疗中有一定作用     

Novel family of gynecologic cancer antigens detected by anti-HIV antibody

Objective: The reactivity of gynecologic cancer proteins with monoclonal antibody (MAb) directed against the human immunodeficiency virus I (HIV-I) was tested.Methods: Cytoplasmic and nuclear proteins, extracted from a broad range of gynecologic cancers obtained during standard surgical procedures, were tested in Western blotting with MAb 5023 developed against the amino acid sequences 308-322 of the envelope protein gp120 of HIV-I.Results: Three cell membrane proteins, M(r)l20,000 (p120), M(r)41,000 (p41), and M(r)24,000 (p24), and one chromatin protein, M(r)24,000 (p24), were detected by MAb 5023 in invasive, poorly differentiated cervical squamous-cell carcinoma; ovarian serous cystadenocarcinoma; poorly and well-differentiated endometrial carcinoma; vulvar squamous-cell carcinoma; and malignant mixed müllerian tumor. The same antigens were identified in cervical carcinoma cell line SiHa. Neither p120 nor p24 was recognized by other MAbs directed against the variable loop of gp120. Antigens p120 and p41 were undetectable in normal ovarian tissue and in biopsy samples of normal vaginal and rectal mucosa. Rectosigmoid cancer as well as colon carcinoma, lung carcinoma, and melanoma cell lines all tested negative.Conclusions: The identified antigens may represent either the products of human genes (proto-onc-ogenes) or, more likely, the products of an unknown virus specifically expressed in female cancer.     

Adverse effects of lupus anticoagulant positive blood sera on placental viability can be prevented by heparin in vitro

OBJECTIVE: Lupus anticoagulant poses a significant risk factor for obstetric complications, whereas heparin improves live birth rates in those pregnancies. Pathophysiology of antiphospholipid antibodies on placental function involves coagulopathies and thrombosis but also dysregulated trophoblast turnover. STUDY DESIGN: With the use of placental explant cultures, we assessed the effect of lupus anticoagulant positive sera (LA + sera) on apoptosis, mitosis, and invasion of trophoblast and determined the role of unfractionated heparin in regulating these functions. RESULTS: LA + sera were associated with increased placental apoptosis (TUNEL, M30 formation, DNA laddering). LA + sera decreased villous trophoblast proliferation and reduced extravillous trophoblast invasion through matrigel. Heparin attenuated LA + sera-induced apoptosis and facilitated trophoblast invasion. CONCLUSION: Lupus anticoagulant may impair placentation by increasing apoptosis, attenuating mitosis and reducing invasion of the trophoblast. The direct effects on trophoblast viability by heparin demonstrate an alternative biologic function for this anticoagulant and raise the possibility that anomalous trophoblast development may be therapeutically regulated.     

Leuprolide acetate stimulates smooth muscle of the human reproductive tract

Leuprolide acetate (10(-4) to 10(-5) M) had a stimulatory effect on spontaneous contractions of human myometrium and fallopian tube in vitro. The uterine and tubal tissues were from patients with dysfunctional uterine bleeding that was treated by hysterectomy. Whether the effect of LA on smooth muscle is a component of the mechanism of therapeutic action or an undesirable side effect remains to be determined.     

Purification of placental alkaline phosphatase and its monoclonal antibody

Monoclonal antibodies (MAbs) to placental alkaline phosphatase (PAlP) were raised by using the hybridoma technique. Spleen cells from immunized mice were fused with the mouse myeloma line NS-1 using 50% polyethylene glycol and cultured in a selection medium. Antibodies were screened by the enzyme immunoassay using immobilized solid-phase antigen and anti-mouse immunogloblin Fab' (rabbit)-beta-D-galactosidase complex. Four double-cloned hybridomas were obtained. The cross-reactivity with different kinds of alkaline phosphatases of the raised MAbs was examined. At first, MAb 11-D-10, with which placenta was stained but liver and small intestine were not stained in indirect immunofluorescence, was selected. Then, the cross-reactivity of MAb 11-D-10 was further investigated by immunoblotting (Western blotting). MAb 11-D-10 reacted with PAlP but did not react with hepatic and intestinal alkaline phosphatase at all. The binding activity of 125I-labeled MAb 11-D-10 with different choriocarcinoma cell lines (SCH, BeWo, NaUCC-1, NaUCC-2, and NaUCC-3) was highest in SCH, then in the order of BeWo, NaUCC-3, NaUCC-1 and NaUCC-2, and correlated to the PAlP content of the cells (SCH greater than BeWo greater than NaUCC-3 greater than NaUCC-1 greater than NaUCC-2). Also the intensity of indirect immunofluorescence of the above cell lines with MAb 11-D-10 correlated with the PAlP content of each cell.     

Evaluation of human sperm-zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens

Characterized WHO monoclonal antibodies (MAbs) to human sperm antigens were evaluated as to whether they inhibited sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the 26 MAbs tested, only one inhibited zona binding. The whole sperm-specific MAb inhibited zona binding by 70%. The MAb also caused strong agglutination. Two procedures, Sephadex column chromatography and papain digestion, were used to determine whether agglutination or steric hindrance was a factor in the capability of MAb to inhibit zona binding. However, inhibition remained comparable to previous results. The MAb did not prevent capacitation, nor calcium influx and the resulting increase in hyperactivated motility and acrosome reaction. Since its inhibitory influence is not due to agglutination factors, steric hindrance or prevention of normal pre-fertilization maturation, the MAb may be blocking a portion of the zona binding receptor and may be useful in elucidating sperm antigens important to sperm-egg interaction. The approach used in this study allows definition of sperm surface antigens involved in zona pellucida binding.     

Monoclonal antibody 1BE12 immunoreactivity with human endometrium. Correlations with hormone receptors and proliferation cell markers.

The monoclonal antibody (MAb) 1BE12 has recently been reported to react with several human normal and abnormal tissues. In human endometrium, it reacts more strongly with carcinomas than with normal tissue. To investigate the effectiveness of MAb 1BE12 in identifying cell proliferation in human endometrial cancers, 1BE12 immunocytochemical assays (ICAs) were performed on frozen (n = 47) and paraffin (n = 100) sections with subsequent computer-assisted microcytophotometric (SAMBA) evaluation of immunoprecipitate distribution. MAb 1BE12 immunoreactivity was not impaired by tissue fixation and paraffin embedding. It reacted with normal proliferative endometrium but not with normal secretory endometrium, and immunoreactivity increased with the degree of cell proliferation and malignancy, the amount of immunostaining being greater in invasive carcinomas than in normal proliferative endometrium and endometrial hyperplasia. ICAs showed no correlation between MAb 1BE12 immunoreactivity and estrogen and progesterone receptor antigenic sites. On the other hand, MAb 1BE12 staining in frozen sections increased with Ki67, EGFR, pHER-2/neu, and cathepsin immunostaining. These findings suggest that ICAs on frozen and paraffin-embedded biopsy specimens using MAb 1BE12 along with other markers can be useful for early detection and grading of endometrial carcinoma. The relevance of MAb 1BE12 to the selection of patients for laser ablation of the endometrium rather than hysterectomy is also discussed.     

The effect of leuprolide acetate on steroidogenesis by granulosa and theca cells in vitro

Enhancement of follicular response to controlled ovarian hyperstimulation for human in vitro fertilization (IVF) has been suggested following pretreatment with leuprolide acetate (LA). However, additional human menopausal gonadotropin (hMG) is required to achieve follicle maturity in the presence of LA. We studied the effect of LA on steroidogenesis of granulosa and theca cells in vitro. Human granulosa cells obtained from IVF follicular fluid aspirations were cultured for 14 days in the presence and absence of human chorionic gonadotropin (hCG). hCG significantly enhanced progesterone (P) and estradiol (E₂) production by the cells, however, the addition of LA in concentrations of 10, 100, and 1000 ng/ml had no effect. Porcine granulosa cells were cultured for 48 hr in the presence and absence of follicle stimulating hormone (FSH) with the addition of LA at the same doses. LA did not affect the FSH-induced increase in P production. Porcine theca cells were cultured for 48 hr in the presence and absence of hCG. The addition of LA did not affect androstenedione (A) production by these cells. We conclude that in this dynamic model in vitro, LA does not inhibit or stimulate P or E₂ production by granulosa or A production by theca cells.     

Monoclonal antibody (5G6.4) against ovarian carcinoma shows inhibition of in vitro colony formation

Monoclonal antibodies (MAbs) have the potential for diagnosis and therapy of cancer, 5G6.4 is a MAb of the IgG2a class which was produced by immunization of BALB/c mice with human ovarian carcinoma (Ov Ca) cells. To further characterize 5G6.4, its effect on cell growth was tested using a human Ov Ca cell line established in our laboratory. A clonogenic assay was set up in 48-well plates in a double agar system. The cells were plated and 5G6.4 was added at different concentrations. Control plates consisted of cells with media without MAb. Negative control plates were also prepared using the same concentrations of an isotype-matched antimelanoma MAb, 225.28s. Colony formation (CF) was reduced to 50% or less of control with increasing amounts of 5G6.4 up to 50 micrograms/ml. Although CF was still depressed at concentrations above 50 micrograms/ml, the inhibition did not follow a directly proportional line; instead, it followed a bell-shaped curve. Plates with the control MAb, 225.28s, did not show this response. Similar results were obtained with cells from malignant Ov Ca ascites in the same clonogenic assay. Our study suggests that in the evaluation of the in vitro effect of MAb on growth, the concentration of MAb is crucial and may not show a linear response and that 5G6.4 may have a direct therapeutic effect by blocking the growth of Ov Ca cells. 5G6.4 is presently under study for therapy in an animal model.     

A monoclonal antibody, 4H12, recognizes a surface antigen found on granulated metrial gland cells in the murine decidua

A monoclonal antibody (MAb), designated 4H12, was selected for reactivity to a surface antigen on PYS-2 teratocarcinoma cells. 4H12 was the product of a fusion of lymphoid cells of a non-immunized pregnant C57BL/6 mouse to NS-1 myeloma cells. Initial studies utilizing immunohistochemistry revealed that MAb 4H12 bound to an antigen found on cells in the decidua basalis of 7-, 8- and 10-day pregnant mice. Antigen-positive cells of 11--19-day pregnant mice were also found predominantly in the decidua. A few antigen-positive cells were found in the labyrinth of the placenta and up against Reichert's membrane. Antigen-positive cells were morphologically and spatially distinct, oval to round with large periodic acid Schiff positive granules. Indirect immunofluorescent (IIF) labeling of decidual cultures showed antigen on the surface of cells that were small, oval to round and adherent. The antigen recognized by MAb 4H12 was removed from tissue sections with trypsin and protease and therefore is suggested to be a protein. We conclude that MAb 4H12 recognizes a surface antigen found on cells historically described as granulated metrial gland (GMG) cells. This MAb should greatly facilitate the further analysis of the life history and function of GMG cells during pregnancy.     

Role of transesophageal echocardiography in detecting left atrial thrombus and spontaneous echo contrast in patients with mitral valve disease or non-rheumatic atrial fibrillation

Instances of left atrial (LA) thrombus and spontaneous echo contrast were evaluated by both transthoracic echocardiography (TTE) and subsequent transesophageal echocardiography (TEE) in 50 patients with rheumatic mitral stenosis (Group I) and 52 patients with non-rheumatic atrial fibrillation (Group II). Among these 102 patients, TEE detected LA thrombi in 16 patients (15.7%) and spontaneous echo contrast in 35 (34.3%). In contrast, TTE revealed LA thrombi in only 8 patients (7.8%) and spontaneous echo contrast in only 2 patients (2.0%). All of the LA thrombi and spontaneous echoes detected by TTE were also found by TEE. When TEE was applied, patients with spontaneous echo contrast had a significantly higher incidence of LA thrombus than did those without this echo (42.8% vs 1.5%, p less than 0.01). Spontaneous echo contrast was coexistent in all but one of the patients with LA thrombi (15 of 16, 93.7%). In Group I, the incidence of spontaneous echo contrast for patients with isolated or predominant mitral stenosis was high (68.3%) when TEE was applied, but this echo was not observed in any patient who had more than a moderate degree of mitral regurgitation. In Group II, 7 patients (13.5%) were also found to have spontaneous echo contrast, which could only be detected by TEE. Of these 7 patients, LA thrombus was noted in 4 by TEE, but only in 1 by TTE. Thus, it can be concluded that: (1) TEE is superior to TTE for detecting LA thrombus and spontaneous echo contrast; (2) spontaneous echo contrast in LA is not only frequently encountered in mitral stenosis without significant mitral regurgitation, but is also found in some patients with non-rheumatic atrial fibrillation; and (3) the presence of spontaneous echo contrast is associated with a higher incidence of LA thrombus and may be considered as a warning sign for further formation of LA thrombus.