Adenovirus-mediated interleukin-24 enhanced the antitumor effect of radiotherapy on nasopharyngeal carcinoma in vitro and in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>AdVIL-24 gene therapy combined with radiotherapy may be a novel and effective treatment strategy for human NPC.</p>

Biocompatibility of physico-crosslinked regenerated silk fibroin film as tissue engineered cornea / 中华实验眼科杂志

Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potentialutility invivo, includingtransparency, mechanicalintegrity, biocompatibilityand slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. ObjectiveThis study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. MethodsHuman corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. ResultsThe morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). ConclusionsPhysico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility.

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.</p>

Suppressive effect of rhIL-24 protein on the human A375 cell melanoma in nude mouse / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the suppressive effect of purified and renaturalized rhIL-24 protein on the human A375 cell melanoma in nude mouse.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Expression of Human Interferon-λ1 and Interferon-ε Gene in WI-38 Cells and Comparison of Their Biological Activity / 中国生物工程杂志

The biological activities i. e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-λ1 and hIFN-ε were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-λ1-His and pcDNA3.1A-hIFN-ε-His by PCR was constructed, then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells ) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFNλ1-His and rhIFN-ε-His, meanwhile MTT assay was used to detect their antineoplastic activities. It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-λ1-His is more powerful than of rhIFN-ε -His. The antiviral molecular mechanisms of both hIFN-λ1 and hIFN-ε are related to MxA. The foundation for further study on the bioactivities and mechanism of action of hIFN-λ1 and hIFN-ε was established.

Expression of Human Interferon-λ1 and Interferon-ε Gene in WI-38 Cells and Comparison of Their Biological Activity / 中国生物工程杂志

The biological activities i. e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-λ1 and hIFN-ε were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-λ1-His and pcDNA3.1A-hIFN-ε-His by PCR was constructed, then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells ) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFNλ1-His and rhIFN-ε-His, meanwhile MTT assay was used to detect their antineoplastic activities. It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-λ1-His is more powerful than of rhIFN-ε -His. The antiviral molecular mechanisms of both hIFN-λ1 and hIFN-ε are related to MxA. The foundation for further study on the bioactivities and mechanism of action of hIFN-λ1 and hIFN-ε was established.

Ad-ING4 inhibits K562 cell growth / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effect of recombinant adenovirus Ad-ING4 on K562 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

The effect of oncolytic adenovirus on human umbilical vein endothelial cell / 生物工程学报

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 effect of human IL-17F on growth of human hepatocarcinoma xenograft tumor in nude mice / 生物工程学报

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 study of the growth-suppression and mechanisms of hepatocelluar carcinoma tumor in nude mice / 生物工程学报

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.

Generation and characterization of antibody against paf1 complex in Drosophila melanogaster / 生理学报

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.

Cellular Apoptosis of C6 Mouse Glioma Cells Induced by hING4 Mediated by Adenovirus / 中国生物工程杂志

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-?B-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported.The C6 cells (mouse glioma cells) were infected respectively with the blank adenovirus carrying GFP (Ad) and the recombinated Ad-hING4-His, then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4-His, were respectively observed by LSCM (laser scan confocal microscope) and transmission electron microscope (TEM), detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell’s apoptosis, which probably is the first one to prove this property of ING4.The experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future was established.

Expression of Human Interferon-?1 and Interferon-? Gene in WI-38 Cells and Comparison of Their Biological Activity / 中国生物工程杂志

The biological!activities i.e. antineoplastic activities and antiviral activity of the two novel kinds of interferons: hIFN-?1 and hIFN-? were studied and compared. First the fusion expression vector: pcDNA3.1A-hIFN-?1-His and pcDNA3.1A-hIFN-?-His by PCR was constructed,then the two kinds of plasmids were transfected into WI-38 (human embryonic lung cells) with liposome. And cytopathic effect (CPE) suppression test was used to study and compare the antiviral activities of rhIFN-?1-His and rhIFN-?-His, meanwhile MTT assay was used to detect their antineoplastic activities.It was found that, antiproliferative activity and MxA protein induction shown by rhIFN-?1-His is more powerful than of rhIFN-?-His. The antiviral molecular mechanisms of both hIFN-?1 and hIFN-? are related to MxA.The foundation for further study on the bioactivities and mechanism of action of hIFN-?1 and hIFN-? was established.

The mitosis and immunocytochemistry of olfactory ensheathing cells from nasal olfactory mucosa / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

The expression of recombinant protein of human interleukin-24 and its anti-tumor mechanism / 生物工程学报

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.

Interferon Stimulating Effect of Ciwujia Eleutherosides or Polysaccharide in S801 Leukemic Cell Cultures / 中国免疫学杂志

S801 leukemic cell line established by our department was used as cells for interferon (IFN) induction and stimulating induction. S801 cell culture of concentration of 5-8x10~6 cells/ml was used for IFN induction and IFN titer of 3.55 Lg IU/ml was obtained by classical procedure. The IFN titer obtained S801 cell cultures after pretreatment with Ciwujia eleutherosides B. D and E or polysaccharide (PES) combined with priming procedure,then using Sendai Virus as an inducer was 10-20 times higher than that in cultures without any pretreatment. These Chinese medical herbs may be the ideal IFN helpinducers. This cell line may be applied for IFN production.