CD44hiCD24lo mammosphere-forming cells from primary breast cancer display resistance to multiple chemotherapeutic drugs.

It has been widely suggested that mammosphere-forming cells from tumor cell lines or primary tumors represent the population of cancer stem cells (CSCs), which is supposed to lead to the failure of routine chemotherapy and the recurrence of the disease. However, it is still difficult to obtain CSCs from primary breast cancer for further investigation. We performed a modified culture system to generate mammosphere-forming cells derived from freshly isolated human breast cancer samples and the breast cancer cell line MCF-7. Cancer stem cell-like phenotypes such as CD44 and CD24 were measured by flow cytometry while alkaline phosphatase (AP) and mammaglobin (MGB1) expression was evaluated immunohistochemically. The expression levels of Klf4, Nanog, Oct4, Sox2 and mdr1 genes were analyzed by quantitative real­time PCR. Resistance to chemotherapeutic drugs was detected through the apoptosis assay upon drug treatments together with the detection of drug-resistant gene mdr1. The results revealed that we successfully obtained mammosphere­forming cells from the primary breast cancer in conditioned medium after 14 days of culture. Mammosphere-forming cells from primary breast cancer displayed a CD44hiCD24lo phenotype as well as positive AP and MGB1 reactivity. Stem cell-related genes such as Klf4, Nanog and Oct4 were detectably expressed in these cells. These cells formed tumor-like structures in the lymph nodes of nude mice, which were morphologically and histologically similar to breast cancer. Compared to the breast cancer cell line MCF-7 or mammosphere-forming cells from MCF-7 cells, the mammosphere-forming cells from the primary breast cancer exhibited resistance to three of four first-line chemotherapeutic drugs investigated through the induction of apoptosis, which was largely associated with the increased expression of drug-resistant gene mdr1 upon drug treatment. In conclusion, mammosphere-forming cells generated from the primary breast cancer exhibit CSC-like properties together with multiple drug resistance. Determination of the sensitivity of these primary cancer-derived mammosphere-forming cells to chemotherapeutic drugs may thus provide useful instructions for individualized therapy against the recurrence of breast cancer in the future.

Enhanced performance of photonic crystal GaN light-emitting diodes with graphene transparent electrodes.

The two-dimensional (2D) triangle lattice air hole photonic crystal (PC) GaN-based light-emitting diodes (LED) with double-layer graphene transparent electrodes (DGTE) have been produced. The current spreading effect of the double-layer graphene (GR) on the surface of the PC structure of the LED has been researched. Specially, we found that the part of the graphene suspending over the air hole of the PC structure was of much higher conductivity, which reduced the average sheet resistance of the graphene transparent conducting electrode and improved the current spreading of the PC LED. Therefore, the work voltage of the DGTE-PC LED was obviously decreased, and the output power was greatly enhanced. The COMSOL software was used to simulate the current density distribution of the samples. The results show that the etching of PC structure results in the degradation of the current spreading and that the graphene transparent conducting electrode can offer an uniform current spreading in the DGTE-PC LED. PACS: 85.60.Jb; 68.65.Pq; 42.70.Qs.

[Effect of Naringin on proliferation and osteogenic differentiation of bone marrow stromal cells in vitro].

PURPOSE: To investigate the differentiation and osteogenic activity of Naringin-induced bone marrow stromal cells (BMSCs) in dogs. METHODS: BMSCs were separated and cultured in vitro and identified by FCM. Then different concentration of Naringin (1×10⁻5, 1×10⁻6, 1×10⁻7, 1×10⁻8 and 1×10⁻9 mol/L) were added to cell culture media to induce BMSCs. The effect of Naringin on BMSCs was evaluated respectively by CCK-8 method and measuring the activity of alkaline phosphatase (ALP). The formation of nodules of calcium was detected by von Kossa staining. The data was analyzed with SPSS20.0 software package. RESULTS: The result of cell-surface marker displayed that the expression of CD34 and CD45 were negative while the expression of CD90 was positive. The values were 0.126%, 0.075% and 95.4%, respectively. Naringin could obviously promote cell proliferation, which exhibited the best effect on proliferation and osteogenic differentiation at concentration of 10-6 mol/L. Calcium nodule (von Kossa) staining was positive. CONCLUSIONS: Naringin-induced bone marrow stromal cells can be differentiated into osteoblasts. Naringin at the concentration of 10-6 mol/L can enhance the proliferation and osteogenic differentiation of BMSCs.

Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans.

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).

The immunologic aspects in advanced ovarian cancer patients treated with paclitaxel and carboplatin chemotherapy.

Till now, little is known about the effects of chemotherapy on the immunity of cancer patients and the ideal timing ("window" period) for immunotherapy combined with chemotherapy. In this study, we addressed the immunogenicity of apoptotic ovarian cancer cells induced by paclitaxel and carboplatin, the immunologic aspects in ovarian cancer patients under chemotherapy, and the CTL response when CD8(+) T cells were stimulated with tumor antigen in the "window" period. The immunogenicity of apoptotic ovarian cancer cells was detected first. Then, blood samples from each ovarian cancer patient were obtained before (S(0)) and at days 5-7 (S(1)), days 12-14 (S(2)) and days 25-28 (S(3)) after chemotherapy. The proportions of immunocyte subsets and the function of NK cells were studied. We found that apoptotic ovarian cancer cells elicited a powerful CTL response with antitumor activity in vitro. The proportions of CD3(+) T cells, CD4(+) T cells and the ratio of CD4(+) to CD8(+) cells did not change significantly on S(1), S(2) and S(3), compared to S(0), whereas the percentage of Treg cells decreased remarkably on S(2). The proportions of Th1, Tc1, CD45RO memory T, NKT cells and the ratio of Tc1 to Tc2 cells increased significantly on S(2). IFN-gamma secreting CD8(+) T cells also increased remarkably on S(2), especially when CD8(+) T cells were stimulated with autologous tumor antigen. From our point of view, chemotherapy induces temporary immune reconstitution and augments anti-tumor immune response. It is probable that the "window" period of days 12-14 after chemotherapy provides the best opportunity for immunotherapy.

Elevated expression of Foxp3 in tumor-infiltrating Treg cells suppresses T-cell proliferation and contributes to gastric cancer progression in a COX-2-dependent manner.

The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients. However, few studies have characterized the regulation of Foxp3 expression and function in Treg cells, which are known to comprise distinct subsets, with different roles in the complex tumor microenvironment. Here, we show that significantly more Foxp3-positive Treg cells accumulated in gastric tumors. In addition, we found increased expression of Foxp3 protein per cell in tumor-infiltrating Treg cells. Moreover, elevated Foxp3 expression in tumor-infiltrating Treg cells was associated with the TNM stage in gastric cancer patients. Importantly, further investigation within the tumor microenvironment showed that expression of Foxp3 in Treg cells correlated with expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Furthermore, Treg cells with higher levels of Foxp3 were able to suppress the proliferation of autologous CD4(+)CD25(-) T cells. The suppression of the effector T-cell response was reversed by COX inhibitors and PGE(2) receptor-specific antagonists. Our data demonstrate a mechanism by which tumor-infiltrating Treg cells with increased Foxp3 expression can mediate immune suppression via COX-2/PGE(2) production in the gastric cancer microenvironment. Thus, we provide new insights into overcoming regulatory T-cell activity, which may be beneficial for the treatment of human gastric cancer.

MR molecular imaging of thrombus: development and application of a Gd-based novel contrast agent targeting to P-selectin.

Molecular imaging of thrombus formation at initial stage requires a robust thrombus-specific contrast agent with high sensitivity. In this study, we report a novel P-selectin-targeted paramagnetic molecular imaging agent and the agent's potential to sensitively detect occult microthrombi on the intimal surface of endothelium. Platelet clots and blood clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that was improved with increasing gadolinium level. In vivo contrast enhancement under part of circulation conditions was assessed in dogs. The micro-thrombi around the femoral vein of dog demonstrated higher signal intensities than the control clots and the adjacent muscle. Histology was performed on regions likely to contain thrombus as indicated by MRI. These results suggest that molecular imaging of P-selectin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of P-selectin and may allow for early, direct identification of microthrombi, leading to early diagnosis.

[Effect of apoptotic ovarian cancer cells induced by paclitaxel-cisplatin on antigen presentation function of DC].

AIM: To explore wheather apoptotic ovarian cancer cells induced by paclitaxel-cisplatin could be cross-presented by antigen presenting cells and promote immune responses. METHODS: DCs were generated from peripheral blood monocytes in RPMI1640 supplemented with GM-CSF and IL-4. After 6 days' incubation, DCs were further co-cultured with either apoptotic HO8910 cell lines induced by paclitaxel-cisplatin or control cells for four hours. Maturation of DCs was compared among different groups according to their surface markers through flow cytometry assay and their phagocytosis ability was evaluated by confocal microscopy scanning assay. RESULTS: Apoptotic ovarian cancer cells induced by paclitaxel-cisplatincan were phagocytized more efficiently by DCs.Resulting is more cellularity and maturation of DCs in apoptotic cell-induced groups than control group (P<0.05). CONCLUSION: Apoptotic ovarian cancer cells induced by paclitaxel-cisplatin exhibit strong immunogenicity, which in turn promote the maturation and antigen presentation of DCs.

CD4(+)CD25(+)CD127(low/-) regulatory T cells express Foxp3 and suppress effector T cell proliferation and contribute to gastric cancers progression.

Increased populations of regulatory T cells (Tregs) impair anti-tumor immunity. Recently, the transcription factor Foxp3 has been reported to play a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. However, Foxp3 is a nuclear protein and is of limited value in the isolation of Tregs, which is a major reason that many functionally relevant aspects of Treg cells are still unknown. Here, we have characterized CD4(+)CD25(+)CD127(low/)- as the surface marker of regulatory T cells in gastric cancer. 88.1-96.1%of CD25(+)CD127(low/-) T cells expressed Foxp3, the frequency of CD4(+)CD25(+)CD127(low/-) regulatory T cells in the peripheral blood of gastric cancer patients was significantly higher than that in healthy controls. Increased CD4(+)CD25(+)CD127(low/-) regulatory T cells were also present in the tumor microenvironment, such as those found in the ascites fluid, tumor tissue or adjacent lymph nodes. Particularly those Treg cells associated with the TNM stage. In addition, we found that CD4(+)CD25(+)CD127(low/-) Tregs suppressed effector T cell proliferation and also correlated to advanced stage of gastric cancer. Thus, CD4(+)CD25(+)CD127(low/-) can be used as a selective biomarker to enrich human Treg cells and also to perform functional in vitro assays in gastric cancer.

Humoral immune responses against tumor-associated antigen OVA66 originally defined by serological analysis of recombinant cDNA expression libraries and its potentiality in cellular immunity.

Immunotherapy for cancer relies on the identification of tumor antigens and efficacy of antitumor immune responses. Serological analysis of recombinant cDNA libraries (SEREX), which is based on the spontaneous humoral responses against potential tumor antigens, has provided a novel strategy for searching novel tumor-associated candidates. Through SEREX analysis, we have identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient. Among these genes, a novel gene, OVA66, was found to be expressed significantly higher in carcinoma samples from cancer patients than in normal controls. Comparing humoral responses to OVA66 between tumor patients and healthy donors, it has been shown that the IgG level against OVA66 was significantly elevated in the serum of cancer patients from different histological types of cancer. To determine whether SEREX-defined OVA66 can trigger promising cytotoxic T lymphocyte (CTL) responses, human leukocyte antigen (HLA)-A*0201-restricted T-cell epitopes were predicted through a computational algorithm. Of four predicted peptides, p306-314 (L235) possesses the ability to induce efficient peripheral blood lymphocyte (PBL)-derived CTL responses capable of specifically recognizing peptide-pulsed T2 cells and lysing carcinoma cell lines expressing both HLA-A2 and OVA66 as determined by cytotoxicity and enzyme-linked immunospot assay (ELISPOT). Taken together, our results demonstrate that the SEREX-defined tumor-associated antigen OVA66 can elicit humoral immunity and may also serve as a potential candidate for T-cell-based immunotherapy for cancer.

Fas Ligand gene transfer enhances the survival of tissue-engineered chondrocyte allografts in mini-pigs.

BACKGROUND AND PURPOSE: Since the Fas/Fas Ligand (FasL) interaction has been recognized as an apoptotic pathway, it eliminates the activated T cells and promotes the survival of grafts. In this study, the effect of FasL transfection of pig chondrocytes on allogeneic transplantation was examined in vitro and in vivo. METHODS: Chondrocytes were isolated from articular and aural cartilages of anesthetized Guizhou Xiang (Gz) pig. The cells were transfected with G418 selected virus, packed from PA317 cells with a constructed plasmid using pig FasL (pGCEN-FasL). The apoptotic effect of FasL transfection was examined on Jurkat cells and activated recipient Gz T cells. The FasL expression was assessed by Western blot and flow cytometry. FasL+chondrocytes-Pluronic F-127 complex was injected into the right abdomen of recipient Gz pig. Histology and morphology of the engineered tissue were examined after 2 and 5 weeks of transplantation. RESULTS: The FasL expression was confirmed in pGCEN-FasL transfected chondrocytes. The expression of FasL of chondrocytes from Gz pig was analyzed by FACS. The apoptosis of Jurkat cells and activated recipient Gz T cells was increased by co-culture with FasL(+) chondrocytes (53.41% and 30.38% (E/T=10:1), in contrast of 32.27% and 13.16% with the control chondrocytes, respectively, P<0.01). FasL(+) chondrocytes-Pluronic F-127 implant expressed FasL and Type II collagen at the 5th week and survived until the 8th week. INTERPRETATION: The result indicates that the expression of FasL by chondrocytes is capable of inducing apoptosis of activated T cells. This suggests a potential role for allogeneic transplantation with chondrocytes.

[Influence of traditional Chinese compound recipes with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice].

OBJECTIVE: To compare the influence of traditional Chinese compound recipes (TCCRs) with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice. METHODS: H(22) cancer-bearing mice were chosen to observe the effects of TCCRs with different efficacy on tumor growth inhibition and detect the proliferation function of T lymphocytes, the activity of natural killer (NK) cells, the changes of T lymphocytes and the content of interferon-gamma (IFN-gamma)and interleukin-4 (IL-4). RESULTS: Tumor weight of H(22) cancer-bearing mice in Yidu Gongdu Recipe (YDGDR, a compound traditional Chinese herbal medicine using poison as an antidote for poison)-treated group was obviously lighter than that in the other TCCR-treated groups and the tumor inhibition rate in YDGDR-treated group was 65.76% (P<0.01). The tumor inhibition rates in other TCCR-treated groups were ranged from 10.1% to 17.1% . Body weight of mice in YDGDR-treated group was obviously decreased and depilation was observed at the same time. Pelage of mice in Fuzheng Peiben Recipe (FZPBR, a compound traditional Chinese herbal medicine for supporting the healthy energy)-treated group grew well, and behavior of the mice was active. Stimulation index (SI) of T lymphocyte transformation in YDGDR-treated group was obviously increased (SI=4.34, P<0.01), which showed the proliferation function of T lymphocyte was very strong. The SI of T lymphocyte transformation in the other groups was less than three, which showed the proliferation function of T lymphocytes was not significant. Compared with normal saline (NS)-treated group, percentages of NK cells in Qinre Jiedu Recipe (QRJDR, a compound traditional Chinese herbal medicine for clearing away heat and toxic substances)-treated, Huxue Huayu Recipe (HXHYR, a compound traditional Chinese herbal medicine for activating blood circulation to dissipate blood stasis)-treated and YDGDR-treated groups were obviously increased and 5.05, 4.07 and 5.17 times more than the NS-treated group, respectively (P<0.01). The activity of NK cells wasn't increased in the FZPBR-treated and HXHYR-treated groups. The production of IFN-gamma induced by T cells in YDGDR-treated group was obviously raised (P<0.05), and the production of IL-4 induced by T cells in QRJDR-treated, HXHYR-treated, Huatan Sanjie Recipe (a compound traditional Chinese herbal medicine for eliminating phlegm and resolving masses)-treated and YDGDR-treated groups was also raised obviously (P<0.01). CONCLUSION: YDGDR has a good effect of inhibiting tumor growth and can reinforce cellular and humoral immune function in tumor-bearing mice. FZPBR can strengthen the body.

[Tumor inhibiting and immunoloregulation effects of Mylabris Mixture on H22 cancer-bearing mice].

OBJECTIVE: To investigate the mechanisms of tumor inhibiting and immunoloregulation of Mylabris Mixture on H22 cancer-bearing mice. METHODS: H22 cancer-bearing mice were chosen to observe the effects of tumor inhibiting and detect the proliferation function of T lymphocytes, the toxicity function of NK cells, the changes of T lymphocytes and the contents of interferon-gamma and interleukin-4. RESULTS: Mylabris Mixture could obviously inhibit the growth of H22 cancer in mice, and the tumor inhibition rat was 65.76%. The stimulation index of T lymphocyte transformation and percentage of NK cells in Mylabris Mixture-treated group were obviously higher than those in the normal control group. The subpopulation proportion of T lymphocytes in Mylabris Mixture-treated group was changed more than the normal control group. The production of interferon-gamma and interleukin-4 by T lymphocytes obviously increased in Mylabris Mixture-treated group (P<0.05, P<0.001). CONCLUSION: Mylabris Mixture has the effect of inhibiting the growth of tumor constitution, and regulating immunological function on mice with tumor. Its mechanisms include the reinforcement of T lymphocyte immune function, NK cell killing function and humoral immune function.

[The effects of tumor antigen gene OVA66 on biological characteristics of tumor cells].

AIM: To explore the effects of tumor antigen gene OVA66 on the biological characteristics of tumor cells. METHODS: The eukaryotic expression vector pcDNA3.1-mock and recombined vector pcDNA3.1-OVA66 were transfected into hepatoma cell line SMMC-7721 by LipofectAMINETM 2000. After stable selection and clone proliferation of cells, the expression of OVA66 gene and protein was detected by RT-PCR, Western blot and immunocytochemical (ICC) staining. The cell growth cycle was analyzed by FACS. The ultrastructure was observed under electron microscope. The experiments in cell migration and invasion were performed in vitro. The OVA66-associated genes were analyzed by genechip technique after gene transfection. RESULTS: OVA66 was highly expressed in 7721 cells transfected with pcDNA3.1-OVA66 at mRNA level. Western blot result demonstrated that OVA66 protein content was notably increased compared with that in control cells. Electron microscope and FACS results indicated that OVA66 gene promoted the growth and proliferation of tumor cells. In vitro, the experiments in cell migration and invasion showed that this gene enhanced migration and invasion of tumor cells. The result of genechip demonstrated that the over- expression of OVA66 gene increased the expression of plasminogen activator inhibitor-1 gene (PAI-1), suggesting that it had some effects on tumor invasion and metastasis. CONCLUSION: Tumor antigen gene OVA66 and it's protein demonstrate a series of biological characteristics of tumor cells, which can promote the proliferation, migration and invasion of tumor cells.

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes.

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and allogeneic HLA-A2.1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.

[Expression of FasL gene in porcine chondrocytes].

AIM: To express the Fas ligand(FasL) gene in porcine chondrocytes. METHODS: The porcine FasL gene fragment was amplified by RT-PCR and then inserted into the pGCEN retroviral vector. The recombinant vector was transfected into packaging cells PA317 which were then screened with G418. Supernatant of screened PA317 cells containing high titer recombinant virus was used to infect porcine chondrocytes. Expression of FasL in chondrocytes was analyzed by FACS and Western blot. RESULTS: The recombinant pGCEN-FasL retroviral expression vector was successfully constructed as shown by restriction enzyme digestion analysis and DNA sequencing. FasL was expressed in 57% of infected chondrocytes. Western blot analysis also confirmed the expression of FasL in chondrocytes. The expressed FasL could induce apoptosis of Fas+ cells. CONCLUSION: The recombinant pGCEN-FasL retroviral vector has been successfully constructed and FasL with biological activity was highly expressed in porcine chondrocytes, which lays the foundation for allogenic chondrocytes transplantation.

[Identification of a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66].

AIM: To identify a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66. METHODS: Dendritic cells (DCs), induced from peripheral blood mononuclear cells(PBMCs) by cytokines, were confirmed by morphological observation and FACS. Mature DCs were pulsed with each of the two synthesized peptides which were selected as possible CTL epitopes by software analysis. The pulsed DCs were used to stimulate autologous CD8+ T cells from an HLA-A2+ healthy donor. One week later, the peptides-pulsed autologous PBMCs were used to stimulate the CD8+ T cells for another 3 times at weekly intervals. The stimulated CD8+ T cells were used as CTLs. The cytotoxicity of CTLs to target cells was detected by lactate dehydrogenase (LDH) release assay and the number of T cells secreting antigen-specific IFN-gamma in CTLs was analyzed by enzyme-linked immunospot assay (ELISPOT). RESULTS: The results of morphology observation and FACS indicated that mature DCs were induced from PBMCs. Of the two peptides, peptide L235(FLPDHINIV) induced peptide-specific CD8+ T cells that lysed HLA-A2+ T2 cells pulsed with L235 and OVA66+/HLA-A2+ SW480 cells. Compared with control peptide, L235 increased the number of IFN-gamma producing T cells. CONCLUSION: This novel OVA66-derived CTL epitope L235 can induce HLA-A2-restrictive CTL response, which lays the foundation for preparation of tumor-specific peptide vaccine.

[Immunological up-regulation of cyclophosphamide-treated mice by turtle blood extract].

AIM: To explore the effect of turtle blood extract on murine immune system. METHODS: We compared the ratio of CD4(+) helper T cells and CD8(+) T cells, NK cell activity, lymphocyte proliferation stimulated by mitogen and capability of cytokine secretion of cyclosphophamide-treated mice with or without treatment of turtle blood extract. RESULTS: Turtle blood extract-treated mice displayed increased CD4(+) helper T cell population, stronger cytotoxicity of NK cells and enhanced of ConA-induced lymphocyte proliferation. In addition, the capacity of IFN-gamma secretion was dramatically up-regulated in turtle blood extract-treated mice group. CONCLUSION: Turtle blood extract can upregulate the immune function of cyclosphophamide-treated mouse.

[Study of HLA molecules and its associated genes expression in human ovarian cancer cells].

AIM: To explore the correlation between the expressions of HLA gene and its related gene, and expression of class II transactivator (CIITA) gene induced by IFN-gamma in human ovarian cells. METHODS: The expression of HLA molecule in the tumor cells was detected by Western blot, immunohistochemical staining and flow cytometry. The expressions of transporters associated with antigen processing (TAP), low molecular weight peptides (LMP), and CIITA genes were analyzed by RT-PCR. RESULTS: Abnormal expression rate of HLA class I molecule reached 45% in the 11 ovarian cancer cells. The expressed abnormalities of HLA class I molecule had relation to those of 4 genes (TAP1, TAP2, LMP2 and LMP7). Expression of HLA class II molecule was identical with that of CIITA gene in the ovarian cancer cells or other tumor cells. After IFN-gamma treatment, expressions of HLA class I and II molecule were increased in the cells of CIITA-expressing constitutively or inducibly, while no enhancement of HLA molecule expression manifested itself in the tumor cells of that CIITA was not yet expressed after IFN-gamma induction. CONCLUSION: Deletion of TAP and LMP gene expression in the ovarian cancer cells is an important factor causing abnormal expression of HLA class I molecule, suggesting that the CIITA gene has involved in the modulation of HLA classes I and II molecule expressions in the tumor cells.

[Cloning, prokaryotic expression of CML66 and preparation of its polyclonal antibody].

AIM: To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody. METHODS: cDNA isolated from the testis using RT-PCR was cloned into pGEMT. After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b(+). The recombinant vector was transformed into BL-21(DE3) through electroporation. 6xHis-tagged CML66 expression was then induced by IPTG. The protein was purified through Ni(2+) affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified protein was injected into rabbits to prepare polyclonal antibody. RESULTS: The cloned cDNA sequence was identical with that previously reported. The target protein was successfully purified. And rabbit's anti-serum with high titer was obtained. CONCLUSION: We have cloned CML66 successfully, expressed and purified the protein in E.coli.Furthermore,rabbit polyclonal antibody has been obtained.