Objective To reconstitute hair follicles in mice using graft chambers,and to study the effect of different cell types on hair follicle regeneration.Methods Full-thickness skin was obtained from the back of C57BL/6 neonatal mice.Then,epidermal cell suspensions were prepared by shredding epidermis after trypsinization,hair follicles and dermal cells were collected by filtration,low-speed centrifugation and density gradient centrifugation,and hair follicle epithelial cells were obtained via trypsinization of hair follicles followed by filtration.Nude mice were classified into four groups to be transplanted with epidermal cells + follicular buds,dermal cells alone,epidermal cells + follicular buds + dermal cells,follicular epithelial cells + dermal cells,respectively.The cells were implanted into the dorsal skin of nude mice using fold chambers.After the grafting,the growth of skin and hairs was observed at the grafted sites on week 1,2,4 and 8,and skin specimens were obtained on week 2,4,and 8 for histological study of hair follicles using hematoxylin-eosin staining.Results After grafting,the chambers on the back of nude mice began to shed with crust formation on week 1; stunted hairs came out and follicle-like structures were seen under the microscope on week 2 at the grafted sites,normal hairs were observed on week 4 and 8 in all the mice except for those transplanted with epidermal cells + follicular buds,and the growth of hairs in mice grafted with epidermal cells + follicular buds + dermal cells and mice grafted with follicular epithelial cells +dermal cells was superior to that in mice grafted with dermal cells alone.Conclusions Hair follicles can regenerate after hair follicle cell transplantation into dorsal chambers in nude mice.Both epidermal cells and dermal cells play indispensable roles in hair follicle reconstitution.

In order to culture the qualified clinicians, we should think about how to improve the quality of clinical practice of obstetrics and gynecology. It is of great importance to emphasize the teachers and students to value the teaching work together. Importance should be attached to the advantage of subject of academy, to make the clinical practice closer to practical, which has a perfect effect and will benefit our work.

Objective To study the effects of Poly(I:C) on lung adenocarcinoma A549 cells viability and illuminate the mechanism of Poly (I:C)-induced apoptosis in A549 cells.Methods A549 cells were transfected with the complex of Poly(I:C) and lipofectamine 3000.The viability of A549 cells was tested by methyl thiazolyl tetrazolium (MTF) method.The apoptotic cells were tested by flow cytometry.The caspase proteins were tested by Western blotting and the expressions of interferon-β (IFN-β) and CXCL-10 were assayed by real-time PCR.After employing the pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK,the variation of Poly (I:C) proapoptosis in A549 cells was observed.RNA interfering experiments were employed to knock down melanoma differentiation related antigen 5 (MDA5) or retinoic acidinduced gene Ⅰ (RIG-Ⅰ),and the above indexes were tested.Results The viability of A549 cells was significantly reduced to 74.92％ ±--6.24％ after 200 ng/ml Poly (I:C) transfection compared with that before transfection (95.32％ ± 3.05％,t =2.883,P =0.041).The apoptotic rates induced by 100,200,400 ng/ml Poly(I:C) were 9.97％-± 0.88％,23.63％-± 1.41％,32.57％-± 2.39％,respectively.All of them were higher than that in the control group (0.74％-± 0.15％),with significant differences (t =4.489,P =0.002;t =11.616,P =0.000;t =16.932,P =0.000).Besides,the death receptor pathway proteins such as TNF-related apoptosis inducing ligand (TRAIL),cleaved-caspase-8 and cleaved-caspase-3 increased obviously.MDA5/RIG-Ⅰ pathway was also activated dramatically and the expressions of IFN-β,CXCL-10 were significantly up-regulated.The apoptotic rates reduced to 3.17％ ± 0.66％,5.35％ ± 0.64％ with pancaspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK pretreatment,compared with the control group (15.87％ ±0.93％),and the differences were statistically significant (t =8.643,P =0.001;t =6.824,P =0.002).Moreover,the expressions of TRAIL,IFN-β and CXCL-10 induced by Poly (I:C) were inhibited with MDA5 or RIG-Ⅰ depletion.Conclusion Poly(I:C) can reduce the survival rate of A549 cells and promote the apoptosis mainly by activating the death-receptor pathway mediated by MDA5/RIG-Ⅰ probably,which may involve in IFN-β,CXCL-10.

BACKGROUND:Bone marrow mesenchymal stem cel s are likely to repair renal injury by differentiating into renal parenchymal cel s. OBJECTIVE:To explore the effect and mechanism of bone marrow mesenchymal stem cel s in the renal repair after mesangial proliferative glomerulonephritis. METHODS:Thirty Sprague-Dawley rats were randomized into normal group, model group and treatment group (n=10 per group). Model group and treatment group were treated with tail vein injection of mouse anti-rat monoclonal antibody Thy1.1 to prepare mesangial proliferative glomerulonephritis models. One week after modeling, rats in the treatment group were given 2×106 bone marrow mesenchymal stem cel s via the tail vein, and rats in the other two groups were given the same volume of normal saline. Two weeks after transplantation, urinary protein, urea nitrogen, creatinine levels were detected;hematoxylin-eosin staining was used for observing pathological changes of the renal tissue under microscope;and the expression of transforming growth factor beta 1 was detected by immunohistochemistry method. RESULTS AND CONCLUSION:The levels of urinary protein, urea nitrogen and creatinine as wel as the expression of transforming growth factor beta 1 in the renal tissue arranged in descending order were listed as fol ows:model group>treatment group>control group, and there were significant differences among three groups (P<0.05). In the model group, diffuse glomerular hyperplasia was observed with the presence of increased extracel ular matrix, partial glomerular sclerosis, and interstitial infiltration of inflammatory cel s;in the treatment group, glomerular hyperplasia, mesangial proliferation and inflammatory cel infiltration were al mitigated compared with the model group. Therefore, bone marrow mesenchymal stem cel transplantation may contribute to renal repair after mesangial proliferative glomerulonephritis, by inhibiting overexpression of transforming growth factor beta 1 in the kidney.

OBJECTIVE:To prepare folic acid(FA)-loaded vincristine(VCR)nano liposome(VCR-nLip-FA)and to study its effects on human liver and lung cancer cells. METHODS:VCR-nLip-FA was prepared by ammonium sulfate gradient method,and particle size,Zeta-potential,encapsulation rate and release rate were investigated. Taking human liver cancer HepG2 cells and lung cancer A549 cells as example,uptake rate and inhibitory effect in vitro (5-80 μg/ml) were compared between VCR-nLip-FA and VCR-nLip. RESULTS:The particle size distribution,average particle size,average Zeta-potential,average encapsulation rate and 24 h accumulative release rate of VCR-nLip-FA were 98.1-159.0 nm,132.2 nm,-40.1 mV,(86.6±3.5)%(n=4)and(42.2± 2.6)%. Compared with VCR-nLip,there was no statistical significance in uptake rate of A549 cells to VCR-nLip-FA and inhibitory effect of VCR-nLip-FA on A549 cell viability (P>0.05);uptake rate of HepG2 cells to VCR-nLip-FA and inhibitory effect of VCR-nLip-FA on HepG2 cell viability increased significantly (P<0.01),in dose-dependent manner. CONCLUSIONS:Prepared VCR-nLip-FA can target anti-tumor drug to HepG2 cells efficiently,and highly inhibit the growth of HepG2 cells. But it has no higher effects on A549 cells.

Aim To study the mechanism of cucurb-itacin E ( CuE )-induced autophagy in HeLa cells. Methods Improved MTT assay was adopted to meas-ure the effect of CuE on cell proliferation. Western blot was used to determine the phosphorylation levels of downstream signaling proteins of mTORC1 and the ex-pression of autophagy associated proteins. ResultsCuE inhibited the proliferation of HeLa cells in a dose-dependent manner, and the 24-h IC50 of CuE was 4. 01μmol· L-1 . CuE significantly inhibited the phospho-rylation of p70 S6 K in a time-and dose-dependent man-ner as evidenced by decreased phosphorylation levels of
the mTORC1 substrate. Meanwhile, the expression of LC3-II, a marker for autophagosome formation, was elevated by CuE treatment, and was further increased in the presence of chloroquine. Furthermore, CuE re-duced the levels of p62/SQSTM1 . These results indi-cated that CuE induced autophagy in HeLa cells. The decreased levels of phosphorylated ULK1 S757 were posi-tively correlated with autophagy induction in HeLa cells. Conclusion CuE is likely to induce autophagy through inhibiting mTORC1 activity.

Objective To compare the efficacy and the side-effect of three different ways in treating the patients with malignant pleural effusion. Methods 98 patients histologically proved malignant pleural effusion were randomly divided into three groups, bleomycin group(BLM), bleomycin with mycobacterium group( BLM + UTL) and blemycin with intertleukino2 ( BLM +IL). 31 patients were treated with bleomycin intrapleural injection in BLM group,32 patients were treated with bleomycin and Utilin's(mycobacterium) intrapleural injection in BLM + ULL group and 35 patients were treated with bleomycin and intertleukin-2 intrapleural injection in BLM + IL group. The therapeutic efficacy, change of performance and side effects were compared among the three groups after one period of treatment. The changes of CEA and TNF in the pleural effusion were examined before and after treatment. Results The therapeutic efficacy and performance improvement were higher in BLM+UTL and BLM+IL group than that of BLM group(P＜0. 05) ,the pleural CEA of post-treatment in three groups were lower than that of pre-treatment(P＜0.01) ,the CEA after treatment in BLM+UTL group and BLM+IL group was lower than that of BLM group(P＜0. 01,respectively). The pleural TNF of post-treatment in BLM+UTL and BLM+IL groups was higher than that of pre-treatment(P＜0. 01 ) in BLM group. The pleural TNF of post-treatment in BLM+UTL and BLM+IL group was higher than that of BLM group ( P＜0. 01 ). Conclusion Intrapleural injection of mycobacterium with bleomycin or interlekin-2 with bleomycin has better efficacy than using bleomycin only in treating malignant pleural effusion.

OBJECTIVE:To prepare Puerarin polymeric micelles and establish a method to determine its entrapment efficiency. METHODS:Puerarin polymeric micelles were prepared by film dispersion method. The polymeric micelles and free drug were sepa-rated by centrifugal-millipore filter filtration method. The entrapment efficiency of puerarin polymeric micelles was determined by HPLC. Diamonsil C18(2)column was used with 1% citric acid solution-methanol(65∶35)at the flow rate of 1 ml/min. The detec-tion wavelength was set at 250 nm,and column temperature was room temperature. RESULTS:The prepared polymeric micelles were spherical and spherical-like in shape with a mean particle size of 54.12 nm,polydispersity index of 0.122,Zeta potential of -13.60 mV;the linear range of puerarin was 2-10μg/ml(R2=0.999 4)with average recovery rate of 99.2%(RSD=0.9%,n=3). The re-covery rate of free drug was 95.3%(RSD=1.7%,n=3). The mean entrapment efficiency and drug-loading amount of puerarin were(35.5±2.12)% and(0.3±0.07)%,respectively(n=3). CONCLUSIONS:Film dispersion method is suitable for the prepara-tion of Puerarin polymeric micelles. Established method is convenient,accurate and reliable for the content and entrapment efficien-cy determination of Puerarin polymeric micelles.

Objective:To evaluate the immunomodulating effect of oyster peptide on immunosup-pressed mice.Methods:ICR mice injected with cyclophosphamide (CTX)were adopted as the module group,with mice without treatment as the control group,and different dosages of oyster peptide (0.5 g/kg,1 .0 g/kg,and 2.0 g/kg)were given to the low,middle,and high groups for 1 5 days.The body weight,spleen,and thymus weight of the mice,structures under the microscope of the immune organs, numbers of white blood cells,ratios of T lymphocyte subsets,immune cytokines and numbers of nuclear cells,and DNA content in bone marrow were all assessed.Results:Compared with the control group, the structures of thymus and spleen of the mice in the CTX group appeared obscure and shrunk when ob-served under microscope,the number of their white blood cells declined (P =0.04),the proportion of their CD3 +T cells in peripheral blood declined (P =0.003),the proportion of their CD8 +T cells in pe-ripheral blood declined (P =0.002),the concentration of their IL-5 in peripheral blood significantly in-creased (P <0.01 ),the concentration of their nucleated cells and DNA density in bone marrow de-creased (P =0.04,P <0.01 ).Oyster could improve the structures of thymus and spleen of the immuno-suppressed mice.Compared with the CTX group,the number of white blood cells in 2.0 g/kg group in-creased (P =0.003),the proportion of CD3 +T cells in peripheral blood in 1 .0 g/kg group (P =0.04) and 2.0 g/kg group (P =0.02)increased,the proportion of CD8 +T cells in peripheral blood in 2.0 g/kg group increased (P =0.002),the concentration of IL-5 in peripheral blood in all the oyster treated groups increased (P <0.01 in 0.5 g/kg,1 .0 g/kg,and 2.0 g/kg groups),the concentration of IL-1 7 in peripheral blood in 2.0 g/kg group decreased (P =0.03),the concentration of nucleated cells in bone marrow of all the oyster treated groups increased (0.5 g/kg vs.CTX,P =0.04;1 .0 g/kg vs. CTX,P =0.02;2.0 g/kg vs.CTX P =0.01 ),the DNA content in bone marrow of all the oyster treated groups increased (P <0.01 in the 0.5 g/kg,1 .0 g/kg,and 2.0 g/kg groups).Conclusion:Oyster peptide could improve the structures of immune organs of the CTX-induced immunosuppressed mice,re-cover the imbalances of T lymphocyte subsets,improve the immune cytokines and increase numbers of nucleated cells and DNA content in bone marrow,thus improving the immunologic function.

Objective To study the radiosensitization of irisquinone on the Warburg effect of MDA-MB231 cells.Methods MDA-MB231 cells in the exponential growth phase were divided into 6 groups:control group,irisquinone group,radiation group,irisquinone plus radiation group(Irisquinone + RA),negative control group,and experimental group (siRNA).Colony formation assay was used to measure cell survival fraction of MDA-MB231 cells.Single-hit multi-target model was used to fit the survival curve and calculate the sensitive enhancement ratio (SER).Flow cytometry was used to measure cell apoptosis.The relative expression levels of HK Ⅱ mRNA and protein in each group were detected by qRT-PCR and Western blot respectively.Results The values of D0,Dq and SF2 in the irisquinone plus radiation group were obviously lower than those in the radiation group.The SER of irisquinone was 1.52.Compared with the other groups,the cell apoptosis rate was increased (t =13.29,12.09,5.90,3.83,P ＜ 0.05),while the relative expression levels of HK Ⅱ mRNA (t =9.14,10.48,3.40,P＜0.05) and protein (t=13.39,16.08,5.81,P ＜ 0.05) were decreased in the irisquinone plus radiation group significantly.Conclusions Radiosensitization function of irisquinone inhibits the Warburg effect of MDA-MB231 cells by down-regulating the expression of HK Ⅱ.