Objective To study the method of obtaining a large number of dendritic cells （DC）. To study the specific cytotoxicity T lymphocyte (CTL) effect against tumor cells initiated by DC pulsed with peptide of cancer cell. Methods Development of cells with cytologic features of DC in bone marrow cultures supplemented with granulocyte macrophage-colony stimulus factor (GM-CSF) and IL-4. Determining the DC phenotype and the specific structure by electronic microscopy. The CTL effect against pancreatic carcinoma leading by the DC pulsed with tumor cells lysate in vitro was observed. Results A large number of typical DC was proliferated by supplementing with GM-CSF and IL-4 cytokines. DC had specific cell appearance and structure, and highly expressed various cell surface molecules. TNF-α had the ability of stimulating DC mature, the mature DC had the enhancing abilities of antigen presenting and IL-12 self-secreting, as well as, expressed higher levels of CD54， MHC-Ⅱ and CD86 molecules than control group (P＜0.05). T lymphoid cell stimulated by DC without tumor antigen could not recognize and kill the target cells, only if DC pulsed with peptide of cancer cell can lead a b immune response to special tumor cells. The inhibiting ratio of CTL was significantly higher than that in other groups (P＜0.01). Conclusion Bone marrow DC has b ability of inducing special CTL against determined cancer cells after they are pulsed with tumor cell lysate. DC vaccine is probably a new immunotherapeutic method against tumor in the near future.
Objective To investigate the feasibil ity of replacing urinary epithel ial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft (BAMG). Methods Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were appl ied to steril ized BAMG to obtain a issueengineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week. Results Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto steril ized BAMG to obtain a tissue-engineered mucosa. Good compatibil ity of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibil ity with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibil ity with BAMG and the compound graft could be a new material for urethral reconstruction.
To study the feasibil ity of human adipose derived stem cells (ADSCs) in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering. Methods The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 × 105/cm2. Cellswere cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02%EDTA treatment, PDGF-BB (50 ng/mL) and TGF-β1 (5 ng/mL) were added at the passage 1 to enhance the smooth muscle cells’ phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry (FACs) was used to examine the positive rate of induced team. Results Cocultured in M-199 media including TGF-β1 and PDGF-BB, the prol iferating capabil ity of the induced cells was significantly downregulated compared with the uninduced cells(P lt; 0.01). The induced cells exhibited “Hill and Valley” morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-l ike morphology. The results of immunofluorescence indicated that the induced cells expressed smooth muscle (SM) cell- specific markers including α-smooth muscle actin (α-SMA), SM-myosin heavy chain (SM-MHC) and Calponin. The results of RT-PCR revealed that the induced cells also expressed α-SMA, SM-MHC, Calponin and SM-22α.The positive rates of α-SMA, SM-MHC and Calponin in FACs were 3.26% ± 1.31%, 3.55% ± 1.6% and 4.02% ± 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% ± 8.31%, 45.33% ± 10.68% and 39.13% ± 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells(P lt; 0.01). Conclusion The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.