Feasibility and Short-Term Stability of Portal Vein Infusion Port Placement by Transjugular Access.

Six pigs underwent implantation of a portal vein infusion port by transjugular access. The technical success rate was 100% (n = 6), with no surgical complications or deaths. At 1 month after implantation, the catheter tip had moved from the splenic vein to the main portal vein, while the catheter protruded into the right ventricle through the right atrium in all cases. Hence, the infusion port system has not been used in clinical practice due to its obvious displacement after implantation. However, this study provides a new idea for future exploration of portal vein infusion pathways.

[Quantitative monitoring of multi-donor chimerism after multi-donor allogeneic hematopoietic stem cell transplantation].

This study was aimed to establish a model for detecting the donor chimerism rate following the multi-donor hematopoietic stem cell transplantations, and simplify its calculation method. Patients with hematologic disease receiving allogeneic hematopoietic stem cell transplantation including single-donor and multi-donor were selected in this study and the donor cell chimerism rates were detected, using STR-PCR combined with capillary electrophoresis. The results indicated that the peaks of the sister alleles coming from the same individual were confirmed to have the approximate areas and can be replaced each other in the situation of mixed chimerism. In the calculation model, the value between reference chimerism and approximate chimerism have no significant difference using the hypothetical peak areas, and the result was confirmed to be accepted basing on typical measurement error between sister alleles (5% - 20%). It is concluded that the areas of share peaks can be replaced by non-share peaks and this conclusion can be used to calculate the double-donor CHM (DD-CHM)(%). Compared to the D alleles, R alleles show more strategic importance because it can lead to more accurate result and allowed simplifying the arithmetic calculations for DD-CHM(%).

[C-kit, NPM1 and FLT3 gene mutation patterns and their prognostic significance in 656 Chinese patients with acute myeloid leukemia].

OBJECTIVE: To evaluate the prevalence and distribution of C-kit, NPM1 and FLT3 gene mutations in patients with acute myeloid leukemia (AML), and to analyze the relationship between the gene mutations and their prognosis. METHODS: Mutations in exon 8 and 17 of C-kit gene, exon 12 of NPM1 gene, exon 20 of FLT3-TKD gene, and exon 14/15 of FLT3-ITD gene were detected by direct sequencing. Clinical data was collected and followed up if the patient had accepted treatment in our hospital. RESULTS: Among the 656 AML patients, mutations in C-kit exon 8 were found in 6 patients (0.9%), C-kit exon 17 in 33 (5.0%), NPM1 in 169 (25.8%), FLT3-TKD in 46 (7.1%), and FLT3-ITD in 178 (27.1%). Six subtypes of mutations were detected in C-kit exon 8, 8 in C-kit exon 17, 11 in FLT3-TKD, 15 in NPM1, of which 5 were not reported before. C-kit exon 17 mutations were more frequently detected in patients with t(8;21) and exon 8 in patients with inv(16) cytogenetic abnormality. No other gene mutations except FLT3 were detected in M(3) patients. NPM1 and ITD mutations were often detected in individuals with normal cytogenetics or M(5) and M(1) of FAB classification, and accompanied with high white blood cell counts in peripheral blood, high blast counts in bone marrow and low CD34 expression. The older the patients were when diagnosed, the more gene mutations and the higher white blood cell count were detected. More mutations were found in individuals with normal karyotype than that with other karyotypes. It appeared that FLT3-ITD was significantly associated with shorter overall survival (OS) (P = 0.004), NPM1 was not significantly associated with OS, but NPM1(+)/ITD(-) patients had the longest OS. CONCLUSIONS: Our results showed that the mutation types and amounts had particular distribution in MICM subtypes, and were associated with white blood cell counts in peripheral blood, blast counts in bone marrow and prognosis. Especially for patients with normal karyotype, the genetic mutations could be new molecule marker.

[The effect of transgenic cell strains expressing recombinant adenovirus vector of hOSM gene on the proliferation and homing ability of umbilical cord blood CD34(+) hematopoietic stem/progenitor cells].

AIM: To establish the transgenic cell strains expressing recombinant adenovirus vector of human Oncostain M(hOSM)gene which is supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34(+) hematopoietic stem/progenitor cell (HSPC) and compare its migration capacity before and after amplification in vitro. METHODS: Establish the transgenic cell strains expressing recombinant adenovirus vector of hOSM gene, and the objective gene was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34(+) HSPC separated by magnetic-activated cell sorting (MACS) was detected by the FCM. After culturing with feeder layer cells, detect the rate of proliferation by flow cytometry (FCM). To compare the homing ability of HSPC after amplification in vitro, detect the spontaneous migration rate and migration rate induced by SDF-1 using transmembrane migration assay (Transwell experiment). RESULTS: The green fluorescence was observed by fluorescence microscope in the transgenic cell strains, and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34(+) HSPC separated by MACS could reach(96.8 ± 2.28)%. After culturing with feeder layer cells for 7 days, the CD34(+) cells were 15.73 times in group containing hOSM more than in group without hOSM. The expression rate of adhension molecules on the surface of CD34(+) cells were also higher in the group containing hOSM than without hOSM. After using Transwell assys to detect the homing ability of culturing cells, the induction migration rate of stem cells clturing on transgenic cell strains was (40.68 ± 1.35)%, significantly higher than the control, which reveals a better homing ability. CONCLUSION: Recombinant adenovirus vector of hOSM gene as feeder layer cells can effectively proliferate umbilical cord blood CD34(+) HSPC in vitro and delay it differentiate, what's more, the stem cells retain a high homing ability after culturing on transgenic cell strains in vitro.

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.

[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.

[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.

[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 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.

[Effect of anti-Helicobacter pylori ureB monoclonal antibody on platelet aggregation and activation, and its mechanism study].

OBJECTIVES: To study the effect of monoclonal antibody (McAb) against helicobacter pylori (Hp) ureB, 1F11 on platelet aggregation and activation, and its mechanism. METHODS: The relativity between human platelet glycoproteins (GPs) and Hp ureB was identified by Western blot and FCM. Platelet aggregation was measured by turbidimetry, and P-selectin and TXB2 assay by ELISA. RESULTS: 1F11 could bind to platelet GPIIIa, and ADP-induced platelet aggregation was inhibited by 1F11 in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 induced by ADP. The FCM results show that the positive rates of platelet binding to FITC-SZ21 was decreased from 99.5% to 77.4% after addition of 1F11. CONCLUSION: McAb against Hp ureB 1F11 inhibits platelet aggregation through binding to platelet GPIIIa but does not block platelet activation. There might be crossed-epitopes on Hp ureB and platelet GPIIIa, and Hp infection might be involved in ITP immunopathology.

[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.