BACKGROUND: Based on the characteristics of cartilage tissue, such as consisting of single type of cells, the cartilage cells or chondrocyte, absence of blood vessel, rather low consumption level of oxygen and nutrition, low level of allo-immunocompetence and simple function in vivo, it seems to be easy for cartilage cell lines to be established for tissue and cell transplantation. We want to set up a cell line with the purpose of current use in tissue engineering in vitro. It will provide the basis for artificial tissue and organ that will become to be standardized and yielded in batch.OBJECTIVE: To explore the potential stimulatory effects of basic fibroblast growth factor(bFGF) and insulin on the proliferation and differentiation in primary culture mice chondrocytes in vitro. The effect and application of the cell factors will be evaluated for tissue engineering.DESIGN: A grouping controlled and repeated trial was conducted with the cells as the subjects.SETTING: Key laboratory of tissue transplantation and immunology of a college.MATERIAIS: The experiment was completed in the Key Laboratory of Tissue Transplantation and Immunology of the Ministry of Education, Jinan University from November 2002 to May 2003. Cultured cartilage cells at random were obtained as the study objects.METHODS: Mice cartilage cells were cultured in medium at the minimum concentrations of serum. The effects of different concentration of bFGF and insulin on the proliferation and differentiation in mice cartilage cells were observed with WST1 and immunofluorescence staining.MAIN OUTCOME MEASURES: Primary results: ① Effect of bFGF on proliferation of primary cultured mice cartilage cells. ② Effect of insulin on proliferation of primary cultured mice cartilage cells. Secondary results:morphological observation of cartilage cells RESULTS: Primary cultured mice cartilage cells were cultured in medium at the minimum concentration of serum(4 g/L fatal bovine serum). It was found that bFGF and insulin might play an important role on the proliferation and growth of mice cartilage cells in a dose-dependent manner. In addition, morphological observation of cartilage cells showed that both bFGF and insulin not only promoted the proliferation of the cells but also enhanced the matrix secretion of cartilage cells.CONCLUSION: Both bFGF and insulin can stimulate the proliferation of cartilage cells in vitro.

Objective: To observe the effect of Trifoliumpratense Leguminosae extract (TLE) on mouse allogenetic skin transplantation. Methods: Recipient BALB/c was divided into physiologic saline (PS) group and TLE group, full-thickness skins were transplanted through back to back method from donor C57BL/6. The allogenetic transplanted skin growth condition was observed. The proliferation of lymphocytes of recipient mice in vitro were detected by CFDA-SE stain and mixed lymphocyte reaction respectively. Results: The allogenetic transplanted skin injected with TLE 25g/kg per day by vena caudalis growed better than that in PS group. The proliferation of lymphocyte in TLE group was smaller than that in PS group. Conclusion: TLE maybe participate in the regulation of mouse immune system and induce its tolerance to the allogenetic transplanted skin.

Aim To investigate the effects of paclitaxel(PTX) on the expression of CD69, CD25 and proliferation of T cells by polyclonal stimulas in vitro, and explore the molecular mechanism of paclitaxel. Methods Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the express of CD69 and CD25 by activated T cells in vitro in response to Concanavalin(Con A) and Phorbol 12,13-dibutyrate(PDB) or T cell proliferation index stained by CFDA-SE in response to PDB+Ion or Con A. Results Paclitaxel had no effect on the expression of CD69, but inhibited the expression of CD25 in activated T cells in response to Con A or PDB in a concentration-dependent manner. Paclitaxel caused a dose-dependent suppression of T cell proliferation to Con A as well as to PDB+Ion. Whether added at the beginning or after 24 h of stimulation by Con A or PDB+Ion, paclitaxel had identical effects. Conclusion The mid and later activation and proliferation of murine T cells stimulated by Con A or PDB+Ion were significantly inhibited by paclitaxel, suggesting that paclitaxel acts on the downstream signaling pathways of PKC?,and not act on the intitial activated associated proteins such as PTK and PKC?.

AIM: In order to understand the pathological changes, characteristic of degeneration in optic nerve and retina after strike of optic nerve. METHODS: According to methods of Allen's spinal injury, a 600gcm-strike power was put on the intraocular portion of the optic nerve and created a striking injury on optic nerve. After a survival interval of 48 h, 1 week, 2 weeks, 4 weeks, 3 months, the animal's optic nerves and retinas were collected and fixed for morphological examination. RESULTS: Forty-eight hours after nerve injuries, the optic nerves were slight enlargement and vacuolation. In 1 week, the optic nerve began to degenerate in injured part and the glia cell had proliferated, but the forms of retinal ganglion cells(RGCs) were normal. In 2 weeks, the vacuolation and focal necrosis were appeared between nerve fiber. The number of RGCs began to decrease. Condensed nuclei presented in the retina. In three month, the diameter of the optic nerve decreased in injury part and collo-scar was formed. The phenomenon mentioned above was more obviously. The internal nuclear neurons and outer nuclear neurons appeared rare. The thickness of retina decreased. The number of RGCs began to decrease in 48 hours and progressed thereafter. It decreased about 3.35%, 13.23%, 19.74%, 23.20%, 29.28% in 48 h, 1 week, 2 weeks, 1 month, 3 months compared with the number of normal RGCs. RGCs began to apoptosis in 48 h. CONCLUSION: The model in this experiment could make definite uncompleted optic nerve and retina injuries. The degree of neuron injuries decreased from RGC, internal nuclear neurons to outer nuclear neurons. The number of RGCs began to decrease in 48 hours, and most quickly periods from 48 hours to one week.

AIM: To establish an animal model for studying the development and metastasis of melanoma. METHODS: C57BL/6 mice were used as host to receive melanoma cell transplantation. Three kinds of melanoma cell lines, B16F0, B16F1 and B16F10, cultured to prepare the cell suspensions, were transplanted into the mouse anterior chamber (AC) of the eye. The time of eyeball diabross, time of survival and metastasis of lymph node and lung were observed. RESULTS: The time of eyeball diabross in F10 group was earlier than that in other groups. The time of eyeball diabross was no difference between F0 and F1 groups. Metastasis was developed 18 days after transplantation in F1 and F10 groups, where the tumor cells was found in ipsilateral cervical lymph nodes. The melanoma cells metastasized to lung in all three groups 28 days after transplantation. The survival time in F0 group was longer than F1 and F10 groups. There was no difference in survival times between F1 and F10 group. CONCLUSION: The differences of three kinds of melanoma cell lines in tumor development and metastasis provided the evidence that was useful for choosing suitable animal model further to study the eye melanima.

AIM: To investigate protective effect of N-acetylcysteine (NAC) on liver and lung in mice after hepatic ischemia/reperfusion injury. METHODS: BALB/c mice were used in a model of partial hepatic ischemia/reperfusion (I/R) injury.They are divided randomly to sham-operated control group(SH), hepatic I/R group or NAC pretreated in hepatic I/R group(I/R-NAC).The level of TNF-α in protal vein and plasma ALT were measured at 1hour and 3 hour, respectively after reperfusion.Lung tissue wet-to-dry(W/D) weight ratio compared. RESULTS: Lung tissue W/D ratio showed significant difference between two groups; The expressions of TLR2/4 mRNA in liver and lung increased obviously after hepatic I/R injury. Histological evaluation showed several changes in lung tissue in I/R group.The level of TNF-α and ALT in protal vein increased continually in I/R group at 1hour and 3 hour of reputation compared with SH group.The level of TNF-α and ALT declined significantly in the group pretreated by NAC. CONCLUSION: N-acetylcysteine can inhibit the activation of TLR2/4 and reduce TNF-α secretion resulted from I/R injury it might abate liver and lung injury following partial hepatic ischemia-reperfusion in mice.

AIM: To investigate the potential of murine epidermal stem cell (ESC) differentiation after seeded in a biodegradable carrier and implanted subcutaneously into syngeneic recipient mice. METHODS: ES cells were induced in vitro to differentiate into ESCs. After stained with a fluorescent dye Hoechst 33342, these ESCs were seeded into a polyglycolic acid (PGA) net containing collagen gel, functioning as a cell carrier, and implanted subcutaneously into 129/J mice, which were syngeneic to these stem cells. RESULTS: The ESCs kept alive in the implant when observed under a fluorescent microscopy 3 weeks or longer after implantation, and could differentiate into hair follicle - like structure,glandular structure, and gave rise to additional structures displaying features resembling native dermis. No apparent rejection or severe side effects were observed at least 10 weeks post- implantation. CONCLUSION: It is feasible to use these ESCs as seed cells in the study to fabricate dermal equivalent having the potential to develop dermal appendages.

BACKGROUND: Endothelin(ET) -1 is a peptide with potent actions on blood vessels and nerve system. Its expression increases in the central nervous system(CNS) in a variety of pathological conditions, inducing harmful effects on the nervous tissue. However it is not clearly elucidated whether the over-expressed ET-1 can directly induce neuronal apoptosis.OBJECTIVE: To investigate whether ET-1 can directly induce apoptosis in primarily cultured brain neurons of rat, and which ET receptor subtype(s) is involved in this action.DESIGN: Completely randomized and controlled experimental study based on cells.SETTING: Neurological department in a university hospital, pathological department of a university and laboratory center of tissue transplantation and immunology, life science and technology college.MATERIALS: This study was completed in the Pathology Department, the Institute of Tissue Transplantation and Immunology, the Life Science and Technology College of Jinan University. The subjects were primarily-cultured neurons obtained from cerebral cortex of newborn rats that were provided by the Experimental Animal Center of the Medical College, Sun Yat-sen University.INTERVENTIONS: After culturing for five days, the neurons were treated with ET-1 (0. 2 nmol/L and 20 nmol/L) for 24 hours. Apoptotic neurons were semi-quantitatively measured with Annexin V and Hoechst 33258 staining respectively. ET-1(20 nmol/L), with BQ123(a selective antagonist for ET receptor A, 1 mmol/L) or with BQ788(a selective antagonist for ET receptor B, 1 mmol/L), was added respectively into the cultures simultaneously. And the apoptotic neurons were quantitatively measured with flow cytometry 24 hours later. Equal amount of PBS, instead of ET-1, waw added into the control subjects.MAIN OUTCOME MEASURES: The effect of ET-1 on apoptosis rate of cultured rat cortical neurons, and the ET receptor subtypes involved in this action.RESULTS: Twenty-four hours after treated with 0.2 nmol/L ET-1, the Annexin-V, and Hoechest 33258 positive stained cell rates[ (23.00 ± 9.96)%,(9.82 ±0.95)% ] were of no difference as compared with those of the controls[ (13.50 ± 3.35)%, (8.21 ± 2. 17)% ]. By contrast, after incubation with the higher dose of ET-1 (20 nmol/L), significant higher rate of apoptosis was measured in Annexin V staining[(50.50 ± 10.78)%, P=0.01, n=4] and Hoechest 33258 staining[(13.78±1.52)%, P= 0. 000, n = 8] . Analyzed with flow cytometry, the apoptosis rate was (0.20±0. 15)% in the control group, (26. 11 ±3.28)% in 20 nmol/LET-1 group, and(13.58 ±4. 92)% in BQ123 +ET-1 and(9.99 ±3.30)% in BQ788 +ET-1 respectively, indicating that BQ123 and BQ788 partially-blocked the apoptosis effect of ET-1 on. cultured neurons(BQ123 + ET-1 vs ET-1, P = 0. 005; BQ788 + ET-1 vs ET-1, P = 0. 001, n = 4, respectively).CONCLUSION: The higher dose of ET-1 (20 nmol/L) can directly induce apoptosis of primarily-cultured cerebral neurons of rats. The effect of ET-1 inducing neuronal apoptosis may be mediated via both ET receptors A and B.

BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity, which has an extensive application in gene therapy for many diseases. However, the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN, TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology, and Department of Pediatric, the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy, natal infant. Informed consent was obtained from his parents, and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate, mean fluorescence intensity of FAM marked CpG ODN, expressions of MNCs, proliferations of lymphocytes and the IgG levels of culture supematants were detected by flow cytometry, fluorescence techniques, MTT assay and ELISA, respectively. MAIN OUTCOME MEASURES: The uptake rate, the mean fluorescence intensity of CpG ODN by MNCs, subgroups and proliferations of lymphocytes, and IgG levels of culture supematants. RESULTS: Compared to the pure CpG ODN group, the uptake rate of the conjugated complexes group was higher (98%), the peak level of up-taking occurred earlier, and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+, CD22+, and CD20+ was increased, A value and IgG levels in supematants were all higher than that of the control group. CONCLUSION: CD40 tigand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte, enhancing its immunological efficiency.

AIM: To investigate the effects of 17?-estradiol (E 2) on the maturation and immunologic function of dendritic cells from human peripheral blood. METHODS: Cultured DCs were treated with E 2 at doses of 10 -7 mol/L and 10 -6 mol/L. The morphologic changes were observed under the scanning electronic microscope. The immunophenotype of DCs in control and treated groups was analyzed by flowcytometry. IL-12 production in culture supernatant was examined by ELISA assay. The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by incorporation of 3H]-TdR. RESULTS: Compared with control group, DCs cultured in the presence of E 2 displayed less dendritic pseudopod, expressed low levels of MHC-II, CD40, CD80 and CD86, and exhibited weakly activity in stimulating the proliferation of allogeneic T cells and reduction of IL-12 production. CONCLUSION: E 2 exerts a negative effect on the maturation and immunologic function of dendritic cells from human peripheral blood.