Objective To explore whether alternatively activated macrophages (aaMphi) can transdifferentiate into lymphatic endothelial cells (LEC) under the inducement of VEGF-C, and to investigate the possible mechanisms involved in aaMphi-induced lymphangiogenesis. Methods An aaMphi model constructed by treating mouse macrophage cells RAW264.7 with mouse recombinant IL-4 for 24h was treated with different concentrations of recombinant mouse vascular endothelial growth factor-C (VEGF-C). After the clustering formed and the tube-like structures were detected in the matrigel, the aaMphi transdifferentiation system was finally decided to be constructed with the VEGF-C in the concentration of 100ng/ml. The mRNA expression of LEC specific markers, VEGFR-3 and Prox1, and aaMphi specific marker, Fizz1, were detected by real time quantitative RT-PCR on the 0, 7th, 14th, and 28th day, respectively, after VEGF-C stimulation. Formation of tube-like structure in the matrigel was observed with inverted phase contrast microscope in 28 consecutive days. Results The VEGF-C induced transdifferention system, incubated in EBM-2 medium and sustained by the matrigel, was successfully established. In this system, it was found that the mRNA expression of VEGFR-3 and Prox1 gradually increased, whilst that of Fizz1 decreased. The mRNA expression of VEGFR-3 and Prox1 reached the peak value, whilst that of the Fizz1 went down to the nadir, on the 14th day. No significant difference in values was found between the 14th day and the 28th day. During the period of the 7th day to the 28th day, distinct tube-like structures were gradually formed in the matrigel and the numbers increased in a time-dependant manner. Conclusion VEGF-C can induce the transdifferentiation of aaMphi into LEC by up-regulating the mRNA expression of VEGFR-3 and Prox1 in aaMphi, which is one of the possible mechanisms involved in aaMphi-induced lymphangiogenesis.

Objective To investigate the effects of rapamycin on renal tissue and function of rats with protein overload nephropathy and to explore the protective mechanism of losartan. Methods Experimental rat models with protein overload nephropathy, induced by intraperiotoneal injection of BSA (2. 0 g/d)into female Wistar rats, were divided into three groups: control group, rapamycin group(injected intraperitoneally with rapamycin) and losartan group(injected intraperitoneally with rapamycin and given orally with losartan). At different time points, the quantity of 24-hour urine protein and renal function were measured, and the morphologic changes of renal tissues were evaluated by HE staining and electron microscope. Results Both at day 7 and day 14, rats received BSA developed intense proteinuria. At day 7, compared with control group, 24-hour proteinuria increased markedly in rapamyein group (P<0.05). Nevertheless,proteinufia was notably alleviated in losartan group (P<0.05). At day 14, 24-hour-urine protein of rapamycin group was also significantly higher than that of the losartan group (P<0.05), but therewas no significant difference between control group and losartan group (P>0.05). Proteinuria and intratubular albumin cast formation were alleviated notably in losartan group. The fusion of focal podocytes in rapamycin group was obvious in comparison with control group. Conclusions Rapamycin can agrravate proteinuria in rats with protein overload nephropathy through changing the barrier of glomerular filtration by damaging podocytes. Furthermore, losartan can alleviate severe proteinuria induced by rapamycin.

Objective To explore the protection and mechanism of hydrogen sulfide (H2S) preconditioning against injury induced by cisplatin in human marrow mesenchymal stem cells (HMMSCs).Methods HMMSCs were treated by cisplatin at 0,5,10,20,40,60,80 mg/L concentrations for 24 h respectively and were exposed to cisplatin at 20 mg/L concentrations for 0,6,12,24,36,48 h respectively.HMMSCs were pretreated with NaHS at 0,0.4,0.6,0.8,1.0 mmol/L respectively for 30 min before exposed to cisplatin at 20 mg/L concentrations for 24 h.HMMSCs were treated by U0126 or combined with human epidermal growth factor (HEGF) together for 30 min before they were preconditioned with NaHS for 30 min.The cell survival rate,cell apoptosis rate,the expression of p-ERK1/2 and t-ERK1/2 were recorded.Results The cell survival rate decreased to 71.72％±2.72％,59.41％±5.44％,50.37％±4.55％,38.97％±2.92％,30.11％±4.64％ and 21.71％±5.35％ respectively after cells were treated with cisplatin at 5,10,20,40,60,80 mg/L concentrations respectively,and the differences were statistically significant compared with 0 mg/L group.The cell viability fell to 70.30％±6.20％,61.63％±2.70％,51.29％±3.13％,38.72％±3.66％ and 27.57％±2.32％ after HMMSCs were treated with cisplatin at 20 mg/L for 6,12,24,36,48 h respectively,and the differences were statistically significant compared with 0 h group.The cell viability increased to 65.99％±2.67％,72.93％±5.44％,75.10％±4.71％ and 76.56％± 5.25％ when HMMSCs got pretreatment of NaHS,and the differences had statistical significance compared with cisplatin group.The cell apoptotic rate decreased from 35.29％±2.77％ to 18.62％±0.97％ when HMMSCs were pretreated with NaHS at 0.6 mmo/L.Treatment of HMMSCs with cisplatin at 20 mg/L for 24 h reduced p-ERK1/2 expression.The pretreatment of NaHS could inhibit the inhibitory action to the expression of p-ERK1/2 induced by cisplatin.Pretreatment with U0126 or HEGF inhibited or promoted the protection and the upregulated expression ofp-ERK1/2 caused by NaHS pretreatment.Conclusion The preconditioning of H2S can protect cell damage caused by cisplatin via activating the ERK1/2 pathway of HMMSCs.

Objective To develop the hypothesis ‘saturated or non-saturated cytotoxicity model' and explain the various phenomena of antibody mediated immunoresponses in recipients,including rejection and accommodation.Methods The imitating complement dependent cytotoxicity.The threshold set to identify as saturated or non-saturated cytotoxicity depends on antigen-antibody complex(R)whether or not above lethal number(D)in effective time.Feasibility of the hypothesis was examined through explaining various phenomena mediated by anti-donor antibodies,especially some contradictory phenomena.Results Hyperacute rejection,accelerated rejection and acute rejection could be well explained by saturated cytotoxicity.Accommodation of ABO imcompatible transplantion,de novo antibody induced injury,change of protein profile,and C4d deposition in graft could be well elucidated by the hypothesis.Conclusion The hypothesis saturated or nonsaturated cytotoxicity model' help to interpret and interconnect various phenomena of antibodies mediated immune response,such as rejection and accommodation.

Objective To investigate the protective effect of hydrodynamics-based injection with plasmids of IL-10, TGF-β1 and TGF-β1 + IL-10 in murine skin transplantation model. Methods Plasmids were constructed by inserting coding sequences of IL-10 and TGF-β1. In F1 mice (Balb/c×C57BL/6, H-2b/d) to Balb/c (H-2d) murine skin transplant model, 20 μg plasmid (blank or IL-10 or TGF-β1 or IL-10 + TGF-β1) was injected to donors by hydrodynamics-based method in first day and every interval 2 days for 6 times. The survival of grafts was observed after 7 days of transplantation. After C56BL/6 spleen cells transfused Balb/c accepted 5 times hydrodynamics-based injection as above,CD4+ CD25+ T regulatory cells of spleen were measured by FACS. Results The survival time of graft in each group was (13.50±1.04)days (blank group), (13.83±1.16)days (IL-10 group), (15.33±1.50) days (TGF-β1 group), and (21.33±3.20) days (IL-10 + TGF-β1 group),respectively (P<0.05). The percentage of CD4+ CD25+ cells was (6. 58±1.86)% (blank group),(10.52±1.13)% (IL-10 group),(14.44±0.42)% (TGF-β1 group),and (14.25±1.24)% (IL-10+TGF-β1 group) respectively (P<0.05). Conclusion Hydrodynamics-based transfection of IL-10 combined with TGF-β1 can synergistically enhance the percentage of CD4+ CD25+ T cells and prolong the graft survival.

Objective To investigate the expression of survivin in T lymphocytes that were stimulated by Con A and alloantigens in renal grafts in vitro and in vivo. Methods According to the different treatments, the experiment was divided into three parts. (1) The C57BL/6 mice splenocytes stimulated by Con A (10 mg/L) were cultured in the RPMI-1640 medium. Following the proliferation blockade or not, the expression of Survivin in the splenocytes was detected. (2) The GVHR model was established by transfusing the C57BL/6 mice splenocytes into Balb/c× C57BL/6 F1 mice, and the expression of Survivin in the donor splenocytes was detected at the different time points. (3) Seventythree cases of clinical renal allograft biopsy specimens were collected, pathologically diagnosed and classified according to the Banff 97 classification, and then the expression of Survivin was detected.Results Survivin was expressed in the CD3+ splenocytes that received Con A stimulation. The positive cell count reached the peak on the day 3, and subsequently declined. In the GVHR model, the lymphocytes infiltration and Survivin expression were detected around the portal vein and portal area on the post-splenocytes-transfused day (PSTD) 4 to 12. But on the PSTD 14, the Survivin expression could not be detected in the infiltrated lymphocytes. In the renal allograft biopsy specimens,lymphocytes did not express Survivin in 13 specimens of the group without acute cellular rejection.was difference between the two groups (P＜0. 01 ). Conclusion The activated T cells possess the capacity to express Survivin, and the expression is time-dependent. For the characteristics of Survivin expression of T cells, it may be applied as an approach to diagnose the acute cellular rejection and judge its degree and stage in the clinical allograft biopsy specimens.

Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.

Objective To investigate treatment and prognosis of patients with osteosarcoma of the extremities.Methods A total of 311 patients with osteosarcoma of the extremities,who had undergone treatment in our institute from 1998 to 2008,were enrolled in this retrospective study.Kaplan-Meier survival curve and Cox regression model were used to analyze the correlation between survival rate and variables including patients' demographics,chemotherapy,surgery,complications,and tumor metastasis.Results Among 311 patients,there were 206 males and 105 females,aged from 5 to 56 years (average,18.6 years).A total of 282 patients underwent aggressive or radical surgery,including 149 cases of limb salvage surgery and 133 cases of amputation surgery.One hundred and five patients underwent standard chemotherapy and 206 patients underwent non-standard chemotherapy.The 5-year survival rate was 57.4％ in patients treated with standard chemotherapy,36.3％ in patients treated with non-standard chemotherapy,16.8％ in patients with lung metastasis,50.7％ in patients without lung metastasis,56.6％ in patients who underwent limb salvage surgery,31.8％ in patients who underwent amputation surgery,44.6％ in patients with Enneking stage Ⅱ B and 33.1％ in patients with Enneking stage Ⅲ.For patients treated by amputation surgery,because non-standard chemotherapy which was performed in most of them and other confounding factors,the 5-year survival rate of them was lower.The Cox regression analysis showed that lung metastasis and non-standard chemotherapy were associated with inferior outcomes.Conclusion Neoadjuvant chemotherapy combined with aggressive or radical surgery could cure about 60％ of patients with osteosarcoma of the extremities.Lung metastasis and non-standard chemotherapy are risk factors that severely affect prognosis.

OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.

AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .