Adenovirus-mediated ING4 expression reduces multidrug resistance of human gastric carcinoma cells in vitro and in vivo.

Chemotherapy is the primary treatment for both resectable and advanced gastric carcinoma, yet multiple drug resistance (MDR) of gastric carcinoma remains a significant therapeutic obstacle. The development of novel strategies to reduce MDR in gastric carcinoma would yield a better outcome following chemotherapy. ING4, a member of the inhibitor of growth (ING) tumor-suppressor family, possesses antitumor and radiosensitization or chemosensitization effects in a variety of human cancers. The present study investigated the effects and possible mechanisms of action of adenovirus-mediated ING4 (AdVING4) on the reversion of human gastric carcinoma cell MDR in vitro and in vivo in nude mouse xenografts. The data showed that the expression of ING4 mRNA and protein was dramatically downregulated (or lost) in gastric carcinoma SGC7901/CDDP cells after CDDP-induced MDR phenotype and in the parental SGC7901 cells. AdVING4­induced ING4 expression reversed MDR and induced apoptosis of SGC7901/CDDP cells in vitro and in vivo in the SGC7901/CDDP xenograft tumors. Furthermore, AdVING4 substantially downregulated the expression of MDR-related proteins P-gp and MRP1 and apoptosis­related proteins Bcl-2 and survivin, but upregulated the expression of apoptosis-related protein Bax in the SGC7901/CDDP xenograft tissues. The reversion effects elicited by AdVING4 on gastric cancer cell MDR were closely associated with the downregulation of ATP-binding cassette transporters and activation of apoptotic pathways. Thus, these findings suggest that AdVING4 may be a feasible modulator for the MDR phenotype of gastric carcinoma cells.

[Adenovirus-mediated interleukin-24 enhanced the antitumor effect of radiotherapy on nasopharyngeal carcinoma in vitro and in vivo].

OBJECTIVE: To investigate the inhibitory effect of adenovirus-mediated interleukin-24 (AdVIL-24) in conjunction with ionizing radiation on the growth of CNE-2Z human NPC cells in vitro and in vivo and underlying mechanisms. METHODS: The CNE-2Z cells were transfected with AdVIL-24 alone or combined with radiotherapy. The transfection efficacy of AdVIL-24 in CNE-2Z cells was determined by RT-PCR and Western blot. Cell growth was assessed by MTT assay, apoptosis was detected by flow cytometry through double staining of cells with propidium iodide (PI) and the expressions of P21, P27, cyclin E, CDK2, Bax, Bcl-2 and caspase-3 were detected with semi-quantitative RT-PCR and western blot, respectively. Anti-tumor effect of AdVIL-24 was observed using CNE-2Z human nasopharyngeal carcinoma transplanted tumor in athymic nude mouse model. The volume and weight of the xenografted tumors were measured and the expressions of P21, P27, cyclin E, CDK2, Bax, Bcl-2, caspase-3, CD34 and VEGF and the microvessel density in xenografted tumors were determined by immunohistochemistry analysis. RESULTS: AdVIL-24 plus radiotherapy induced cell growth inhibition (P < 0.05, Q3d, 4d = 0.916, 1.050) , cell cycle G1 phase arrest(50.37%, P < 0.05, Q = 1.042) and apoptosis (48.82%, P < 0.05, Q = 1.042) , substantial up regulations of P21, P27, the ratio of Bax/Bcl-2 and cleaved caspase-3 and down regulations of cyclin E and CDK2 (P < 0.05, QP21 = 0.959, QP27 = 0.956, Qcyclin E = 1.078, QCDK2 = 1.046, QBax/Bcl-2 = 0.995) in vitro. In the xenografted tumors, AdVIL-24 plus radiotherapy induced cell growth inhibition (P < 0.05, Qvolume14 = 1.053, Qweight = 1.004) , apoptosis (P < 0.05, Q = 0.974) , substantial upregulation of P21, P27, the ratio of Bax/Bcl-2 and cleaved caspase-3 and downregulation of cyclin E and CDK2 (P < 0.05; QP21 = 0.920, QP27 = 0.937, QcyclinE = 1.060, QCDK2 = 1.019, QBax/Bcl-2 = 0.982, Qcleaved-Caspase-3 = 0.927) , decreased the tumor vessel CD34 expression and microvessel density. AdVIL-24 potentially blocked the radiation-induced enhancement of VEGF. CONCLUSION: AdVIL-24 gene therapy combined with radiotherapy may be a novel and effective treatment strategy for human NPC.

[The in vitro and in vivo effects of adenovirus-mediated inhibitor of growth 4 and interleukin-24 co-expression on the radiosensitivity of human lung adenocarcinoma].

OBJECTIVE: To study the radiosensitivity of the recombinant adenoviral vector (called Ad-ING4-IL-24) carrying and co-expressing inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) to human lung adenocarcinoma and the underlying mechanisms. METHODS: The expression levels of ING4 and IL-24 were detected by Western blot. The growth-suppressing and apoptosis-inducing effect of Ad-ING4-IL-24 combined with radiotherapy on SPC-A-1 lung carcinoma cells were assessed by MTT assay and FCM respectively. The 25 nude mice were randomly divided into 5 groups of 5 mice ecah: PBS group, Ad group, Ad-ING4-IL-24 group, radiotherapy group and joint group (Ad-ING4-IL-24 combined radiotherapy). Mice in all groups except radiotherapy group were intratumorally injected every other day for 6 cycles. The short and long axes of the tumor were measured dynamically, tumor volume was calculated as: V = L × W(2/2), changes in tumor volume were graphed. The human lung carcinoma model was established with SPC-A-1 cells in nude mice. The ratios of tumor-suppression and q were calculated. The expression of Caspase-3, Bcl-2, Bax, VEGF in tumor samples were detected by immunohistochemistry. RESULTS: The expressions of ING4 and IL-24 were successfully expressed in SPC-A-1 cells. MTT assay and FCM showed that the levels of cell-growth inhibition and apoptosis induction in Ad-ING4-IL-24 combined with radiotherapy group [(86.2 ± 0.8)%, (60.9 ± 1.0)%] were higher than in Ad-ING4-IL-24 group [(49.8 ± 0.3)%, (26.3 ± 1.3)%] and in radiotherapy group [(44.4 ± 2.2)%, (33.3 ± 0.8)%] (ratio of cell-growth inhibition, F = 550.88, P < 0.01; ratio of induced apoptosis F = 614.08, P < 0.01). Ad-ING4-IL-24 combined with radiotherapy showed an enhanced radiosensitivity effect on human lung adenocarcinoma (q = 1.20). In Ad-ING4-IL-24 group, radiotherapy group and Ad-ING4-IL-24 combined with radiotherapy group, the weight inhibition ratio was 49.5% (5 nude mice), 35.4% (5 nude mice), 79.8% (5 nude mice) respectively. Ad-ING4-IL-24 combined with radiotherapy had a synergetic and enhanced radiosensitivity effect on inhibiting the growth of transplanted tumor (q = 1.18). According to immunohistochemistry, Ad-ING4-IL-24 was shown to up-regulate the expression of Bax and Caspase-3 but down-regulate the expression of Bcl-2 and VEGF. CONCLUSION: Ad-ING4-IL-24 had an enhanced radiosensitivity effect on human lung adenocarcinoma, and therefore acted as a radiotherapy sensitizer, which may be related to its effect on apoptosis-induction and antiangiogenesis.

Cytocompatibility of regenerated silk fibroin film: a medical biomaterial applicable to wound healing.

OBJECTIVE: To explore the feasibility of using regenerated silk fibroin membrane to construct artificial skin substitutes for wound healing, it is necessary to evaluate its cytocompatibility. METHODS: The effects of regenerated silk fibroin film on cytotoxicity, adhesion, cell cycle, and apoptosis of L929 cells, growth and vascular endothelial growth factor (VEGF) expression of ECV304 cells, and VEGF, angiopoietin-1 (Ang-1), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2) expression of WI-38 cells were assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, viable cell counting, flow cytometry (FCM), and enzyme-linked immunosorbant assay (ELISA). RESULTS: We showed that the regenerated silk fibroin film was not cytotoxic to L929 cells and had no adverse influence on their adhesion, cell cycle or apoptosis; it had no adverse influence on the growth and VEGF secretion of ECV304 cells and no effect on the secretion of VEGF, Ang-1, PDGF and FGF2 by WI-38 cells. CONCLUSION: The regenerated silk fibroin film should be an excellent biomaterial with good cytocompatibility, providing a framework for reparation after trauma in clinical applications.

[Growth inhibition and mechanisms of human bladder cancer T24 cells by adenovirus-mediated ING4 gene in vitro].

OBJECTIVE: To explore the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the human bladder cancer T24 cells in vitro. METHODS: The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of ING4 in human bladder urothelial carcinoma T24 line. The influence of Ad-ING4 transfection on cell proliferation was evaluated by MTT assay and cell apoptosis by Hochest33258 staining and flow cytometry. RT-PCR and Western blot were performed to detect the transcriptional level of such apoptosis-related genes as Bcl-2, Bax, p53, HIF-1α and caspase-3. RESULTS: Human ING4 was successfully transcribed in T24 cell. Apoptosis rate of T24 cell in Ad-ING4 group was significant higher than that in control groups (17.2% ± 4.1% vs 4.7% ± 1.2% and 4.8% ± 1.2%, P < 0.05). Ad-ING4 not only up-regulated the expression of p53 and Bax(1.40 ± 0.11 vs 0.27 ± 0.04, 1.50 ± 0.12 vs 0.60 ± 0.05) and down-regulated the expression of Bcl-2 and HIF-1α (0.19 ± 0.02 vs 1.20 ± 0.08, 0.33 ± 0.03 vs 0.98 ± 0.06, all P < 0.05), but also enhanced the caspase-3 activation and eventually led to apoptosis. CONCLUSION: Adenovirus-mediated ING4 gene exhibits anti-tumor capacity in T24 human bladder cancer cell and induces in vitro apoptosis. It may be related to the up-regulation of P53 and Bax/Bcl-2, and the down-regulation of HIF-1α. Thus the caspase-3 activation is enhanced so as to lead to apoptosis.

[In vitro and in vivo inhibitory effect of Ad-ING4 gene on proliferation of human prostate cancer PC-3 cells].

BACKGROUND AND OBJECTIVE: Adenovirus vector has been widely used in tumor gene therapy. ING4 is a member of growth inhibiting factors and a potent anti-tumor gene which could induce apoptosis of many tumor cells. This study was to investigate the inhibitory effects of adenovirus-mediated ING4 (Ad-ING4) gene on the proliferation of human prostate cancer PC-3 cells in vitro and in vivo, and to explore its mechanisms. METHODS: Ad-ING4 was obtained by virus-amplification technique. After transfection of purified Ad-ING4 into PC-3 cells, the expression of ING4 was detected by reverse transcription-polymerase chain reaction(RT-PCR); the influence of Ad-ING4 transfection on cell proliferation was evaluated using MTT assay. Cell apoptosis was assessed using Hoechst33258 staining and flow cytometry. RT-PCR was performed to detect the mRNA levels of the transcription of apoptosis-related genes such as bcl-2, bax, p53, and caspase-3. Athymic nude mice bearing PC-3 tumors were intratumorally injected with Ad-ING4 (100 microL, 1x10(9) pfu/mL). Tumor growth was recorded. All nude mice were killed at the end of the experiment to observe the growth of xenografts. The expressions of Bcl-2, Bax, Caspase-3, and CD34 proteins in tumor tissues were detected by immunohistochemistry. RESULTS: Human ING4 gene was successfully transcribed in PC-3 cells and induced apoptosis by up-regulating p53, bax, caspase-3 expression and down-regulating bcl-2 expression. Inhibition of cell proliferation was significant in PC-3 cells. Tumor growth was significantly inhibited in the Ad-ING4 group as compared with that in the Ad-GFP group and the PBS group (P<0.05). The weight inhibitory rate was 37.0% in the Ad-ING4 group. The expressions of Bax and Caspase-3 were up-regulated, and the expressions of Bcl-2 and CD34 were down-regulated in the Ad-GFP group. CONCLUSIONS: Adenovirus-mediated ING4 gene exhibits anti-tumor ability in human prostate cancer PC-3 cells in vitro and in vivo, and induces apoptosis. This may be related to the up-regulations of p53, bax, Caspase-3 and down-regulation of bcl-2.

[Construction of adenovirus expressing anti-vascular endothelial growth factor hairpin ribozyme and its inhibition of growth of colorectal cancer HT-29 cells].

BACKGROUND AND OBJECTIVE: Oversexpression of vascular endothelial growth factor (VEGF) is correlated to poor prognosis of colorectal cancer. This study was to construct replication deficient adenovirus carrying anti-VEGF hairpin ribozyme and investigate its inhibitory effects on VEGF expression and growth of colorectal cancer HT-29 cells. METHODS: Anti-VEGF hairpin ribozyme was cloned into the transfer vector pAdTrack-CMV, and recombined with adenoviral backbone vector pAdEasy-1 in BJ5183 bacteria. The recombinant plasmids were packaged and amplified in 293 cells (named Ad-Rz). The expression of anti-VEGF ribozyme in HT-29 cells was examined by RT-PCR. Inhibition of Ad-RZ on VEGF mRNA and protein expressions were detected by real-time PCR and ELISA, respectively. Inhibition of cell growth was measured by MTT assay. HT-29 cell xenografts were established in nude mice and the microvessel density (MVD) was determined by CD34 staining. RESULTS: The anti-VEGF ribozyme was successfully inserted into the adenoviral vector. Anti-VEGF ribozyme was effectively expressed in HT-29 cells. After infection by Ad-RZ, the relative expression of VEGF mRNA was decreased to about(45+/-0.01)% of that of PBS control group in HT-29 cells (P<0.05); the amount of VEGF protein in the supernatant of Ad-Rz treated cells [(OD=(0.46+/-0.35)/million cells] was lower than that of PBS treated cells [(OD=(0.80+/-0.35)/million cells] (P<0.05). Although Ad-Rz did not significantly inhibit cell growth of HT-29 cells (P>0.05), it significantly inhibited tumor angiogenesis of HT-29 cell xenografts in nude mice, compared with PBS group (P<0.05). The proliferation rate of xenografts treated by Ad-Rz was lower than that treated by PBS, but the difference was not significant (P>0.05). CONCLUSION: Anti-VEGF hairpin ribozyme mediated by adenovirus could inhibit the expression of VEGF and the tumor angiogenesis in colorectal cancer HT-29 cells.

[Impact of regenerated silk protein membrane on the cytokine expression of transfected human corneal epithelium cells].

OBJECTIVE: The purpose of this research was to study the influence of the regenerated silk fibroin film (SF) on cytokines expression of transfected human corneal epithelial cells (HCECs) and to investigate the possibility of constructing biomaterial complex using SF, modified by transgenic cells. METHODS: Empirical study.Ad-VEGF(165) was injected into the limbus of a rabbit's cornea to induce cornea neovascularization (CNV). CNV was evaluated by growth areas and VEGF characteristic was evaluated by immunohistochemistry. HCECs was cultivated on silk protein membrane in the cell cultivation plate. Modality of cells, activity of cell proliferation and infection efficiency of Ad-VEGF(165) were monitored to evaluate the biocompatibility of silk fibroin. The angiogenesis-related cytokines in the cell cultivation supernatant was measured using ELISA method such as vascular endothelial growth factor (VEGF), angiogenin 1 (Ang1), epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in the supernatant (Two-way analysis of variance). RESULTS: (1) The area of corneal neovascularization was observed to be (7.60 +/- 1.12) mm(2) at 1 week after Ad-VEGF(165) was injected and it became (12.28 +/- 2.54) mm(2) another three weeks later. Positive expression of VEGF in corneal stromal was observed by immunohistochemistry at 3 d, 1 week and 1 month after injection. (2) There was no difference noticed in amorphous, growth curve and infection efficiency of Ad-VEGF(165) between both cells culture conditions of silk protein membrane and plate cultivation. (3) After transfection, the concentration of VEGF, Ang1, EGF and TGF-beta expressions in the corneal epithelium cell cultivation supernatant with silk protein membrane as carriers was (721.67 +/- 66.97) ng/L, (1042.67 +/- 315.81) ng/L, (2421.00 +/- 0.00) ng/L, and (313.33 +/- 34.06) ng/L respectively; and the concentration of each of the aforementioned expression was (721.67 +/- 66.97) ng/L, (860.33 +/- 315.81) ng/L, (1960.33 +/- 797.90) ng/L, and (278.00 +/- 53.11) ng/L without using silk protein membrane as carriers. The increase of VEGF (F = 168.16, P < 0.0001), EGF (F = 52.76, P < 0.0001), Ang1 (F = 12.47, P = 0.001), and TGF-beta (F = 0.008, P = 0.932) in the Ad-VEGF(165) group was considered statistically significant; however, there was no evident change in the concentration of VEGF (F = 0.071, P = 0.793), EGF (F = 0.563, P = 0.465), Ang1 (F = 0.14, P = 0.714), and TGF-beta (F = 0.008, P = 0.932)expressions in the corneal epithelium cell cultivation supernatant both with or without using silk protein membrane as carriers. CONCLUSION: Same as cell HCECs culturing in the cultivation plate, through SF application, VEGF(165) destination gene could be high-level expressed in the supernatant having which the HCECs is cultured on SF, and in addition, the angiogenesis-related cytokines content of Ang1, EGF, and TGF-beta autocrine in the HCECS cultivation supernatant could be high-level expressed as well.

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth.

BACKGROUND: Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhIL-24) on pancreatic cancer. METHODS: Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n = 10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD). RESULTS: The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0 x 10(10) pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05). CONCLUSION: The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

[Suppressive effect of rhIL-24 protein on the human A375 cell melanoma in nude mouse].

OBJECTIVE: To study the suppressive effect of purified and renaturalized rhIL-24 protein on the human A375 cell melanoma in nude mouse. METHODS: Human A375 cells were injected into the nude mouse. After the volume of tumor attained, rhIL-24 was injected into the tumor. 2 weeks later, the tumors were resected for measurement of volume and weight, following with pathological and immunohistochemistry examination. RESULTS: The volume and weight of tumors were decreased markedly after treatment of rhIL-24, when compared with those in controls. The expression of Bax gene upregulated, while Bcl-2, CD34 and VEGF gene downregulated. It indicated tumor growth inhibition and inducing of apoptosis of tumor cells. CONCLUSIONS: rhIL-24 has a suppressive effect on the A375 cell melanoma in nude mouse. It can also induce the A375 cell apoptosis without side effect on nude mouse.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts.

AIM: To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. METHODS: The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. RESULTS: VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. CONCLUSION: Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

[Ad-ING4 inhibits K562 cell growth].

OBJECTIVE: To observe the effect of recombinant adenovirus Ad-ING4 on K562 cells. METHODS: Human ING4 recombinant transfer vector pAdTrack-CMV-ING4 was constructed by enzyme digest and ligation of human ING4 gene which was obtained through site specific point mutation of mouse ING4. The vector was co-transduced into BJ5183 E. coli with pAdEasy-1. The new recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-hING4 was transfected into QBI-293A cells. To obtain the ING4 recombined adenovirus (Ad-ING4). Ad-ING4 was used to infect K562 cells. The effect on K562 cells of ING4 was tested by LSCM FCM and immunohistochemistry. RESULTS: Human ING4 recombinant adenovirus vector was constructed successfully, and high titre ING4 recombinant adenovirus (Ad-ING4) was obtained. ING4 can down-regulate the expression of bcl-2 and up-regulate expression of bax. The apoptosis of K562 cells induced by ING4 was proved by LSCM FCM and immunohistochemistry. The apoptosis rate was 19.7% (after 72h), which displayed significant difference compared with that of control groups (P < 0.01). CONCLUSION: Ad-ING4 can inhibit the growth of K562 cells and induce the cells apoptosis. The human ING4 recombinant adenoviral vector constructed might provide an approach to the target therapy of tumors.

Generation and characterization of antibody against paf1 complex in Drosophila melanogaster.

Paf1 complex was identified in yeast and characterized to function in transcription and its related events. We identified the Drosophila homological components of paf1, CDC73 and RTF1 of paf1 complex. The genes encoding Drosophila paf1, CDC73 and RTF1 were cloned and expressed. With the purified recombinant proteins of truncated components of paf1 complex, antibodies against the Drosophila paf1, CDC73 and RTF1 were generated. These antibodies have been shown to be able to detect the endogenous paf1 subunits as well as their human counterparts in the HeLa extract. On Drosophila polytene chromosomes, these antibodies have been demonstrated to locate the paf1 complex at actively transcribing sites, which co-localized with phosphorylated RNA polymerase II, indicating that paf1 complex in Drosophila is involved in transcription or the events coupling with transcription.

[The study of the growth-suppression and mechanisms of hepatocelluar carcinoma tumor in nude mice].

Study effect and mechanisms of growth-suppression of hepatocelluar carcinoma (HCC) in nude mice. The construction of the pAdeasy-1-pTrack-CMV-hIL-24 recombined adenovirus vector (Ad-hIL-24) was completed and lineared with PacI. Ad-hIL-24 were transfected into QBI-293 cells and obtained. 16 nude mice of the subcutaneous tumor models were established with SMMC-7721 HCC and were randomly divided into NS, 5-Fu, Ad and Ad-hIL-24 groups. Then 100 microL NS, Ad (10(7) pfu) and Ad-hIL-24 (10(7) pfu) for each one were given respectively QOD, and 5-Fu (20 microg/kg) were injected Q.D., for 5 times, with intratumor injections. After 15 d, 16 mice were sacrificed and subcutaneous tumors were taken out. The volumes (before administration, 1 week and 2weeks after administration) were measured and the weights of tumor were weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor cells were observed under microscope. Caspase3, P53 and P27, CD34 and VEGF were tested in immunohistochemistry. In tumor subcutaneous model, compared with NS group, the ratios of tumor-suppression of Ad-hIL-24 group and 5-Fu group were 68.52% (P < 0.01) and 65.64 (P < 0.01), respectively. Caspase3 protein in Ad-hIL-24 group was higher than other 3 groups significantly (P < 0.01). The expression of P27 also differed from NS group (P < 0.01). CD34 and VEGF protein in Ad-hIL-24 group can inhibit neovascularization obviously (P < 0.001), compared with NS and Ad groups. Ad-hIL-24 inhibits the growth of SMMC-7721 HCC on nude mice's. The mechanisms of tumor-suppression may be multi-pathways such as the induction of caspase3 pathway, P27 activities and the antiangiogenic mechanism.

[The effect of human IL-17F on growth of human hepatocarcinoma xenograft tumor in nude mice].

The human interleukin-17F(hIL-17F) gene was amplified by RT-PCR from PHA-activated human peripheral blood mononuclear cells (PBMCs). It was then subcloned into the retrovirus vector pSIV-1. The pSIV-1/hIL-17F together with its two-helper virus vectors pHIT456 and pHIT60 cotransfected into the package cell 293T by lipofectin to produce mature recombinant retrovirus, which was then used to infect SMMC-7721 hepatocarcinoma cells (HCCs), and the cells were selected in the presence of G418. The integration, transcription, expression of hIL-17F gene in SMMC-7721 cells was identified by PCR, RT-PCR and Western blot respectively. MTT and FCM showed that hIL-17F couldn't alter the proliferation and cell cycle of SMMC-7721 cells, but ELISA showed that it could down-regulate IL-6, IL-8 and VEGF expression. The effect of rhIL-17F supernatant on growth suppressing of ECV304 cells was observed by MTT. The experiment of human hepatocarcinoma xenograft tumor in nude mice showed that the formation and growth rates of hIL-17F-transgenic SMMC-7721 showed an obvious decline, and VEGF and CD34 expression and angiogenesis of the transgenic neoplasms was also evidently defined. hIL-17F can markedly inhibit the growth of human hepatocarcinoma xenograft tumor in nude mice by antiangiogenesis. This study provided an experimental evidence for further conducting tumor gene therapy by targeting vascularity and exploiting antiangiogenic novel medicine related to hIL-17F.

[The effect of oncolytic adenovirus on human umbilical vein endothelial cell].

The E1A gene was obtained by PCR with QBI-293A cell genome DNA as template. After enzyme digestion, the E1A gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-E1A were lineared by PmeI and co-transformed with pAdEasy-1 in BJ5183 E. coli. The recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-E1A were digested by PacI and transfected into QBI-293A cells with liposomes. The oncolytic recombinant adenovirus Ad-E1A was obtained after 7 days. The results showed that this oncolytic adenovirus Ad-E1A can replicate in ECV304 cells and inhibit growth of ECV304 cell. In addition, it also decreased the secretion of VEGF and expression of NF-kappaB of ECV304 cells, indicating that Ad-E1A have potential of inhibition of tumor metastasis.

[The expression of recombinant protein of human interleukin-24 and its anti-tumor mechanism].

The hIL24 cDNA sequence was cloned into prokaryotic high expressive vector pET-21a(+) and recombinant hIL24 was expressed in E. coli with IPTG induction. The purified recombinant hIL24 exhibits following functions in HeLa cell: inhibiting cell growth, inducing apoptosis, inducing PMBC to secrete IL-6, TNF-alpha, IFN-r and inhibiting blood vessel formation. Our preliminary results suggest that the apoptosis induced by rhIL24 is through down-regulating expression of anti-apoptosis factor Bcl-2 and activation of mitochondria apoptosis pathway.

The mitosis and immunocytochemistry of olfactory ensheathing cells from nasal olfactory mucosa.

OBJECTIVE: To culture olfactory ensheathing cells (OECs) of rats in vitro and to investigate its morphology, mitosis and immunocytochemistry, and to explore if the OECs could be a new donation for transplantation. METHODS: OECs were harvested from olfactory mucosa of Sprague Dawley rats based on the differing rates of attachment of the various cell types, followed by glial fibrillary acidic protein (GFAP), nerve growth factor (NGF), anti-low affinity receptor for NGF (NGFRp75), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and S-100 immunocytochemistry. The morphological changes and mitosis were observed under a phase contrast microscope at different culture time. RESULTS: Three morphologically distinct types of cells, bipolar, multipolar and flat morphology were present in the primary culture of adult rat olfactory mucosa. Mitosis was characterized by a retraction of all processes, forming a sphere that divided into spherical daughter cells, the daughter cells sent out their processes. The OECs were immunoreactive for GFAP, NGFRp75, S-100, NGF, BDNF and NT-3. CONCLUSIONS: The OECs from nasal olfactory mucosa cultivated in the medium with fetal bovine serum could survive, divide, differentiate, and express the neurotrophin. It may become an accessible source for autologous grafting in spinal cord injury.

[Combined effects of endostatin gene transfer and ionizing radiation on lung adenocarcinoma model of A549-cells].

OBJECTIVE: The combined inhibition effects of endostatin gene transfer and ionizing radiation on lung adenocarcinoma model of A549-cell were investigated. METHODS: Human endostatin gene was transferred into lung adenocarcinoma A549 cell by retrovirus-mediation to obtain an A549/Endo cell. A549 and A549/Endo cells were xenoimplanted in nude mice respectively (each group included 10 mice). Then, the changes of the tumor size of each group (n = 5) and its growth inhibition rate were estimated. The implanted tumors of each group (n = 5) were exposed to radiation with 20 Gy at 28 day after implantation. They were irradiated again with 20 Gy 3 day later. The ionizing radiation was strictly confined to the tumor by shielding the rest of the body with lead. The size of tumor was measured periodically. The microvessel density (MVD) of 4 groups of implanted tumors (A549, A549/Endo, A549 + IR and A549/Endo + IR) were compared on day 42 postgrafting by the immunohistochemical method. RESULTS: PCR confirmed that endostatin gene was inserted into the genomic DNA of human lung adenocarcinoma A549 cell. The tumor formation time showed significant difference (P < 0.05) between group A549 (7.8 +/- 1.6) d and group A549/Endo (12.2 +/- 1.7) d. At the time of day 42 postgrafting, the tumor sizes of group A549 and group A549/Endo were (927.8 +/- 269.2) mm3 and (217.5 +/- 81.5) mm3 respectively (P < 0.01), and the tumor growth inhibition rate was 76. 5%. At the time of day 14 after irradiation, the tumor sizes of group A549 and group A549/Endo were (157.7 +/- 49.0) mm3 and (4.6 +/- 2.9) mm3 respectively (P < 0.01). The results of immunohistochemical detection showed that the MVD of-A549/Endo implanted tumor was significantly decreased [21.62 +/- 3.55 compared with A549 implanted tumor 35.78 +/- 5.67 (P < 0.01)]. Moreover , it also showed that ionizing radiation could further reduce the MVD of A549/Endo implanted tumor from 21.62 +/- 3.55 to 11.32 +/- 2.78 (P < 0.01). CONCLUSIONS: Retroviruses can highly mediate the transfer of endostatin gene into the adenocarcinoma cells. Endostatin gene transfer can inhibit the xenoimplanted tumor growth by its direct inhibition on neovascularization. The combination of endostatin gene transfer with ionizing radiation treatment can synergistically inhibit the neovascularization and the growth of lung adenocarcinoma.