This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells (BMSCs). IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed under transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ratio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34+ and CD34- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34+ and CD34-,total MNC led to a significant increase in the expansion of CD34+ cells compared with CD34 enrichment (P0.05). These differentiated EPCs were stained positive for CD34+, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34+ and AC133+cells accounted for 68.2%?6.3% (n=6) and 57.2%?9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34+ and CD34- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expanded in vitro and provide seed cells for neural tissue engineering.
Animals, Newborn ; Cell Separation ; Cells, Cultured ; Cerebral Cortex/*cytology ; Culture Media ; Neurons/*cytology ; Rats, Sprague-Dawley ; Stem Cells/*cytology ; Tissue Engineering

The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34- and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P < 0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P > 0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2 +/- 6.3)% of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

BACKGROUND: Infusion of hemopoietic stem cell from donors can promote the chimeric formation and induce specific immunologic tolerance in the allograft recipients. However, the pretreatment for cell transplantation has great toxicity to recipients. So immunosuppressant combined bone marrow infusion is introduced to anti graft versus host reaction. OBJECTIVE: Based on microchimerism, to study the security and associativity of chimera formation induced by kidney-bone marrow transplantation and immunologic tolerance.DESIGN, TIME AND SETTING: The contrast observation was performed at the department of urinary surgery, The Third People's Hospital of Zhengzhou City from January 1998 to December 2005.PARTICIPANTS: According to ABO/Rh blood type and HLA matching, 96 female patients with chronic renal failure and waiting for kidney transplantation were divided into 2 groups, In the combination group, patients received kidney combined bone marrow transplantation; the other uremia patients received the other kidney of cadavers were served as control group. The donors were 48 healthy males. METHODS: Bone marrow of donors was collected simultaneously with kidney obtain and preserved with cryoprotectant at -198 ℃ in nitrogen canister. After kidney transplantation, large dose of anti-human lymphocyte immune globulin were used for 2 weeks, then (0.9-2.5)×10~8/kg mononuclearcell was reinfused. PCR-SRY was used to identify donor derived cell-chimerism. Lymphocyte subgroup of recipients was determined by blood test; and interleukin 10 was measured by enzyme linked immunosorbent assay; in addition, the mass concentration of tumor necrosis factor α and tumor necrosis factor β was detected. MAIN OUTCOME MEASURES: Chimerism, lymphocyte subsets and cytokines were detected at various time points following transplantation. Simultaneously, the transplantation results and complication status of recipients were observed. RESULTS: The positive rate of chimera in the combination group was greater than that of the control group (P < 0.05). The 3-year follow-up showed that incidence differences of acute rejection between recipients with positive chimera and recipients with negative chimera had significance (13%, 35%, P < 0.05). There was no graft versus host disease occurred in the combination group. CONCLUSION: Kidney-bone marrow transplantation can augment chimerism in early postoperative period, and significantly reduce the rate of acute rejection, which is safe and beneficia1to induce specific immunologic tolerance in the renal allograft recipients.

[Objective] In order to explore the optimal minor-factor culture system of CIK/NK cells by proliferating CIK/NK cells using single-factor,bi-factor,and tri-factor combinations of IL-2,IL-7,and IL-12.[Methods] Ficoll-Hypaque method was used to separate cord blood mononuclear cells and divide them into 6 groups.Add single-factor,bi-factor,and tri-factor combinations (IL-7;IL-12;IL-7 + IL-12;IL-2 + IL-7;IL-2 + IL-12;IL-2 + IL-7 + IL-12) of IL-2 (80 ng/mL),IL-7 (40 ng/mL),and IL-12 (40 ng/mL) and culture them in total 21 days then harvest the cells.To collect cell suspensions of each group in culture outset and day 21 and to count the proportion of CD3+ CD56+ CIK cells and CD3- CD56+ NK cells with flow cytometry.[Results] The proportion of CIK cells in single-factor culture system of IL-12 was the highest in all groups (30.23 ± 1.18%).The proportion of CIK cells in bi-factor culture system of IL-2 + IL-12 can raised to 18.58 ± 0.68%.The proportion of CIK cells of the two groups above can reach the level of that using traditional multi-factor culture methods.NK cell proportion in IL-12 was 30.23 ± 1.18%,NK cell proportion in IL-2 + IL-7 can also reach to 29.52 ± 0.89%.[Conclusions] It is adoptable to proliferate CIK/NK cells using minor-factor culture system of IL-12,IL-2 + IL-12,or IL-2 + IL-7.

BACKGROUND:IKVAV-containing peptide sequence is an active region that promotes the adhesion, growth, and differentiation of neural cells in the laminin. It can be fabricated into a novel tissue-engineered scaffold material by self-assembly into hydrogel.OBJECTIVE: This study was designed to synthesize IKVAV-containing (C16H31O-AAAA GGGEIKVAV) peptide. The peptide was observed self-assembly into three-dimensional and porous hydrogel after triggered with phosphate buffered saline (PBS).DESIGN, TIME AND SETTING: Single-sample experiment, performed in the Laboratory of Department of Orthopedics,Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology between September 2004 and January 2005.MATERIALS: IKVAV-containing peptide was synthesized by solid phase method.METHODS: Peptide purity and relative molecular mass were detected by high performance liquid chromatogram and mass-spectrometry, respectively. 1% peptide, whose pH value was equal to 9.5, was self-assembled into hydrogel with the addition of PBS. The ultramicrostructure of the hydrogei was observed through the use of transmission electron microscope (TEM).MAIN OUTCOME MEASURES: Peptide purity and relative molecular mass were detected. Moreover, gross observation of the peptide after self-assembly and TEM observation of peptide ultramicrostructure were performed.RESULTS: Peptide relative molecular mass was 1351.6, which was in accordance with its theoretical value. Peptide purity was 95%. After triggered with PBS, 1% peptide was self-supported into hydrogel in a few seconds. TEM results showed that self-assembled hydrogel consisted of the interconnected nanofibers which varied from 3 to 6 nm in diameter and 100 to 1 500 nm m in length.CONCLUSION: The IKVAV-containing peptide was synthesized and self-organized successfully into porous hydrogel,which was triggered with PBS solution.

Lack of biocompatibility and bioactivity is a big problem for the synthetic materials that have been generated for neural tissue engineering. To get around the problem and generate better scaffold for neural tissue repair, we intended to generate nano-fibers by self-assembly of polypeptide IKVAV. Bioactive IKVAV Peptide-Amphiphile (IKVAV-PA) was first synthesized and purified, the property of which was analyzed and determined by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Then, by addition of hydrogen chloride (HCl), self-assembly of IK-VAV-PA was induced in vitro and nano-fibers formed as shown by transmission electron microscopy (TEM). The effect of IKVAV nanofibers on adherence of PC12 cells was assayed in cell culture and the results showed that the rates of adherence of PC12 increased significantly when the density of IKVAV was within a certain range (0.58 microg/cm2 to 15.6 microg/cm2). However, its effect on the rates of adherence did not significantly alter with time, whether after 1 hour or 3 hours of culture. In general, we showed that IKVAV-PA can successfully self-assemble to form nanofiber, and promote rapid and stable adherence of PC12 cells, and the effect of the self-assembled IKVAV to promote PC12 cells adherence is dosage-dependent within a certain range of densities.

KLD-12 peptide with a sequence of AcN-KLDLKLDLKLDL-CNH(2) was synthesized and its biocompatibility was assessed in animals. Rabbit MSCs were cultured in the hydrogel for 2 weeks. Live cells were counted by using Calcein-AM/PI fluorescence staining. MTT was employed to assess the viability of MSCs cultured in KLD-12 peptide solution of 0.01%, 0.03%, and 0.05%. Hemolysis test, skin irritation test and implantation test were conducted to evaluate its biocompatibility with host tissues. Our results demonstrated that the MSCs in hydrogel grew well and maintained round shape. Cell survival rate was 92.15% (mean: 92.15%+/-1.17%) at the 7th day and there was no difference in survival rate between day 7 and day 14. Cell proliferation test showed that the A value of the KLD-12 solutions was not significantly different from that of control groups (complete culture media) (P>0.05) at the 24th and 48th h. The hemolysis rate of KLD-12 solution was 0.112%. Skin irritation test showed that the skin injected with KLD-12 solution remained normal and the score of skin irritation was 0. The histological examination with HE staining exhibited that the skin layers were clear and there was no infiltration with neutrophilic granulocytes and lymphocytes. It is concluded that KLD-12 peptide hydrogel had a good biocompatibility with host rabbit and MSCs, and KLD-12 peptide hydrogel can provide an appropriate microenvironment for MSCs.

Objective To explore the clinical application value of ultrasound-guided intrahepatic biliary puncture combined with internal double biliary stenting under DSA in elderly patients with malignant hilar biliary obstruction (MHBO).Methods Totally 108 elderly MHBO patients received interventional treatment were analyzed retrospectively.Half of patients were treated with ultrasound-guided intrahepatic biliary puncture (ultrasound group),and another 54 patients were treated with DSA intrahepatic biliary puncture (DSA group).After successful puncture,the patients received percutaneous transhepatic cholangial drainage (PTCD) with 4 methods under DSA guidance,namely internal double biliary stenting,contralateral external PTCD with single biliary stenting,complete external PTCD and external PTCD on the dominant side.The recent complications of intrahepatic biliary puncture at two groups and the curative effect with four methods were observed.Results The frequency of intrahepatic biliary puncture,the dosage of contrast agent,the incidence of pain at the puncture point and hemobilia in ultrasound group were all lower than those in DSA group (all P＜0.05),the successful rate of intrahepatic biliary puncture in first time was significantly higher compared with DSA group (P＜0.05).The liver function indicators at 14 days postoperation and total bilirubin at 21 days postoperation had statistical differences between any two biliary drainage methods (all P＜ 0.05).Conclusion Ultrasound-guide intrahepatic biliary puncture combined with internal double biliary stenting under DSA can significantly benefit elderly patients with MHBO.