Objective To optimize the method of isolating ,culturing and screening fetal mouse liver stem cells in vitro ,and to identify the potential of bi‐directional differentiation .Methods The fetal liver stem cells of mouse were isolated by the density gra‐dient centrifugation and cell difference adherence method ,the proliferation of stem cells was determined by cell plate cloning tech‐nique and MTT method;stem cells were induced for differentiation by adding DMSO and HGF .Results The isolated stem cells showed adherence within 24 h ,which were orbicular‐ovate ,closely packed ,activated within 1~2 weeks ;the positive rates of CD133 , CD49f and EPCAM were (97 .95 ± 1 .21)% ,(92 .71 ± 3 .49)% and (50 .73 ± 3 .45)% respectively ;AFP and CK19 proteins were expressed;red glycogen granules were seen by PAS after induced differentiation;ALB and HNF‐4αwere expressed .Conclusion Fe‐tal hepatic stem cells are successfully isolated by the density gradient centrifugation combined with difference adherence method ,and the isolated cells have strong stemness and proliferation ability ,as well as the ability of bi‐directional differentiation towards hepato‐cytes and bile duct epithelial cells .

BACKGROUND:Long-term use of corticosteroids (hereinafter referred to as hormone) after renal transplantation could obviously lead to adverse reactions. Immunosuppressive regimen with less and no hormone has been a hot focus in the study of renal transplantation al over the world. However, reduction or withdrawal of hormones has a certain risk. At present, there is no unified scheme. Because urine protein can be immediately detected after tubular injury, to monitor urine protein can find the renal dysfunction after transplantation in recipients undergoing renal transplantation, which can gain time for clinical therapy. OBJECTIVE:To discuss the influence of hormone (prednisone) removal on the occurrence of urine protein in recipients undergoing renal transplantation. METHODS:A total of 35 recipients undergoing renal transplantation after removal of prednisone received immunosuppressive regimen of cyclosporine A or tacrolimus+mycophenolate mofetil bivalent. Initial dose of prednisone was 30 mg/d, and then gradual y reduced by 5 mg per week, and withdrawn at 1 month after renal transplantation. There were 16 cases in cyclosporine A group and 19 cases in tacrolimus group. Urine protein was measured and quantified at 3, 6, 12 and 24 months after renal transplantation and 3, 6 and 12 months after addition of prednisone in both groups. Simultaneously, serum creatinine, fasting glucose, body mass increases, the rate of acute rejection, infection, patient/graft survival at 2 years after renal transplantation and urine protein at 24 hours before and after adding hormone were recorded. RESULTS AND CONCLUSION:For the two groups, urineα1-microglobulin started to rise after 6 months of removal of prednisone. Urinary microalbumin, urinaryα1-microglobulin, and urinary transferrin ascended obviously at 12 months. Urinary protein was positive in five cases of cyclosporine A group and in three cases of tacrolimus group. At 24 months, urinary microalbumin, urinaryα1-microglobulin, urinary transferrin and urinary IgG ascended obviously. Urinary protein was positive in cyclosporine A group with 11 cases and in tacrolimus group with 10 cases. 24-hour urinary protein quantity was more than 1 g in every case. On this base, we made the patients to take more prednisone for 6 months, so urineα1-microglobulin and urinary microalbumin began to descend. Each group had one case of positive urinary protein turning to negative. Twelve months after the adjustment of the prednisone, urinary microalbumin, urinaryα1-microglobulin, and urinary transferrin descended respectively. Positive urinary protein turned into negative:in cyclosporine A group with two cases and in tacrolimus group with three cases. 24-hour urinary protein quantity was around 0.7 g. Two years after renal transplantation, serum creatinine and acute rejection rates were higher in the cyclosporine A group than in the tacrolimus group (P<0.05). No significant difference in fasting glucose, body mass increase, infections, and patient/graft survival was detectable between both groups. Results suggested that removal of prednisone greatly affected urine protein in recipients undergoing renal transplantation. In particular, at 2 years after renal transplantation, urinary microalbumin, urinaryα1-microglobulin, urinary transferrin and urinary IgG ascended obviously, and the security needs further research.

Objective To study the apoptosis of human leukemia K562 cells induced by snake venom of Agkistrodon Halys Pallas in Zhejiang Province.Methods IC_ 50 value and cytotoxity of K562 cell were detected by MTT method.Apoptotic cells were dyed by Hoechest 33258.Sub-G1 peak and cell cycle were detected by FCM.Protein expression of Bcl-2 gene was detected by FCM and western-blot method.Results The snake venom of Agkistrodon Halys Pallas in Zhejiang Province inhibited the growth of K562 cells,which appeared dose-dependent.The snake venom induced apoptosis of K562 cells.Meanwhile,protein expression of Bcl-2 was down-regulated.Conclusion Snake venom of Agkistrodon Halys Pallas in Zhejiang could induce apoptosis of human leukemia K562 cells.The mechanism may be related to down-regulation of Bcl-2 gene expression.

Abstract BACKGROUND: Calcium dobesilate (calcium dihydroxy-2, 5-benzenesulfonate) has been widely used to treat chronic venous insufficiency and diabetic retinopathy, especialy many clinical studies showed that calcium dobesilate as vasoprotective compound ameliorates renal lesions in diabetic nephropathy. However, there are few literatures reported calcium dobesilate in the treatment of chronic renal alograft dysfunction after renal transplantation. OBJECTIVE:To observe the efficacy and safety of calcium dobesilate on chronic renal dysfunction after renal transplantation. METHODS:A total of 152 patients with chronic renal alograft dysfunction after renal transplantation were enroled from the Military Institute of Organ Transplantation, Changzheng Hospital, Second Military Medical University of Chinese PLA. They were randomly divided into the treatment group (n=78) and the control group (n=74). Patients in the treatment group received 500 mg of calcium dobesilate three times daily for eight weeks. Al patients were treated with calcineurin inhibitor-based triple immunosuppressive protocols and comprehensive therapies. RESULTS AND CONCLUSION: For patients receiving calcium dobesilate, serum creatinine, blood urea nitrogen and uric acid decreased significantly at two weeks after treatment and maintained a stable level (P < 0.05). However, serum creatinine and blood urea nitrogen returned to the original level soon after drug withdrawal. No significant difference was observed in blood cel count, liver function, blood lipids, electrolytes, blood pressure and 24-hour urine output between the two groups before and after therapy (P > 0.05). Administration of calcium dobesilate did not change the general condition of patients with renal insufficiency, nor did it affect blood concentrations of the immunosuppressive agents. Calcium dobesilate may help to delay the progress of graft injury in patients with chronic renal graft dysfunction by conjugating with creatinine, ameliorating the impaired microcirculation and its antioxidant property. The decline in serum creatinine aleviates patients’ anxiety and concern arising from the elevation of creatinine. However, the negative interference with serum creatinine caused by calcium dobesilate should be cautious in order to avoid misjudgment of patients’ condition.

[Objective] To study the factors relative to the delayed diagnosis of spontaneous dual arteriovenous fistulas (DAVF) and its influence on the prognosis.[Methods] We included 102 continuous patients diagnosed DAVF in the First Affiliated Hospital of Sun Yat-sen University,and analyzed the correlative factors and impact on outcome of diagnostic delay.Outcome was whether symptoms were non-improvement,improvement or restoration at discharge.[Results] Median delay from onset to diagnosis was 3 months (interquartile range,1 to 6).Compared with patients diagnosed carlier(diagnose time≤3 months),patients diagnosed later (diagnose time ＞ 3 months) had a lower frequency of headache (P =0.012),ptosis (P =0.035) and parenchymal lesions (P =0.001),a higher frequency of conjunctival congestion (P =0.004),tinnitus (P =0.021),visual dysfunction (P ＜ 0.001),isolated visual dysfunction (P =0.007) and delayed imaging scan (P ＜ 0.001),a higher frequency of endovascular treatment,and a lower frequency of improvement or restoration at discharge (P =0.033),in which patients with visual dysfunction had a lower frequency of improvement or restoration than those without visual dysfunction (P =0.023).Compared to those with visual dysfunction and other symptoms,patients with isolated visual dysfunction had a higher frequency of onset with paroxysmal blurring or blinding (P ＜ 0.001),two eyes involved (P ＜ 0.001) and more severe visual loss (P =0.057),a higher frequency of draining into transversesigmoid sinus (P ＜ 0.001) instead of cavernous sinus (P ＜ 0.001),and suffered intracranial hypertension all (median intracranial pressure,405 mmH2O;interquartile range,370 ～ 512 mmH2O).However,no statistically significant differences were found in the frequency of improvement or restoration at discharge between two groups (P =0.739).[Conclusion] Diagnostic delay was considerable in this cohort and was associated with outcome,especially in patients with visual dysfunction.

Epidemiological data,clinical observation and animal experiments have shown that cold wave is closely associated with the onset of stroke.When a population with stroke etiology or risk factors is under pre-stroke state,they will have stroke under the influence of various inducing factors.Cold wave is the external factor that causes the body to enter the pre-stroke state and there are many possible mechanisms.The drug intervention of stroke-prone hypertensive rats at 1 week before the cold wave can reduce the occurrence of stroke during cold wave,suggesting that it is of great significance to conduct preventive intervention to the pre-stroke population before the cold wave coming.

BACKGROUND: It is a hot investigation to many scholars that how to cure and prevent renal ischemic reperfusion injury in a utility way, but the mechanism is unclear at present. The investigation indicates that aquaporiin-1 plays an important role during this process. OBJECTIVE: To research the correlation between aquaporin-1 expression and renal function change following renal ischemic reperfusion injury. DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at Histology and Embryology Laboratory of Dalian Medical University from June 2007 to December 2008. MATERIALS: A total of 80 healthy female adult Wister rats were randomly divided into control group and ischemia-reperfusion group. Rats in each group were observed at days 1, 2, 3, 5, and 7 after operation, with 8 rats for each group. The ischemia-reperfusion injury was established on the left kidney. METHODS: Right kidney was removed. The left renal pedicle was freed and occlused to establish ischemia-reperfusion injury model. After 40 minutes, the blood was re-flowed. If the kidney colored from dark red to bright red within 2-5 minutes, the ischemia-reperfusion injury models were successfully established, and the thrombus was not formed in the kidney vessels. If the kidney was still dark red after 5 minutes, the thrombus was formed, and the rats were excluded from the ischemia-reperfusion group. The abdomen was sutured after 40 minutes.MAIN OUTCOME MEASURES: Rats were sacrificed at days 1, 2, 3, 5, and 7 after ischemia-reperfusion injury. Samples of urine, serum, and kidney were performed with the examinations of urine, renal function, renal pathology and morphology, immunohistological assay of aquaporiin-1, and RT-PCR assay. RESULTS: After ischemia-reperfusion injury, the rats had hydrouria, urine osmotic pressure depress, symptoms of carnine and urea nitrogen increasing. HE staining demonstrated that renal tubular epithelial cells were swelling, necrosis, and desquamate. Aquaporin-1 expression and its mRNA level was decreased; in particular, the expression and level were the lowest at day 1 after ischemia-reperfusion injury and recovered to normal value at day 5 after ischemia-reperfusion injury. CONCLUNSION: The down expression of aquaporin-1 maybe one of the important indicators to reflect renal functional changes of acute renal failure following renal ischemia-reperfusion injury.

Objective To increase the understanding in protein-losing enteropathy (PLE).Methods Sixty-one PLE patients were enrolled in the study and the clinical characteristics, complicated disease, diagnosis and treatment were analyzed. Results The age of the patients was 16-77 (40±15)years, and the gender ratio was 35:26 (female: male). The main clinical manifestations were bilateral lower limb edema in 51 cases, ascites in 41 cases, bilateral pleural effusion in 23 cases, pericardial effusion in 13cases, abdominal pain in 16 cases and diarrhea in 33 cases. The prominent abnormality in laboratory examinations was hypoalbuminemia. The underlying diseases include systemic lupus erythematosus (SLE) in 28 cases, intestinal lymphangiectasia in 12 cases, hepatic cirrhosis in 5 cases, heart diseases in 5 cases,Crohn's disease in 3 cases, membranous nephropathy in 2 cases, Budd-Chiari syndrome in 1 case. Four cases happened after abdominal operation and 1 case after radiation therapy of gastric cardia cancer. Thirtyseven cases were diagnosed by 99Tcm-labelled human serum albumin scintigraphy and 24 cases were diagnosed clinically. Treatment was focused on underlying diseases. The clinical manifestations in 21 cases of SLE improved after SLE was controlled. In 2 cases of intestinal lymphangiectasia and one with Crohn's disease, the clinical manifestations improved after surgery. The other patients had no improvement.Conclusions PLE was not uncommon in clinical practice. Its predominant characteristics were severe hypoalbuminemia, edema and dropsy of serous cavity. PLE can complicate other diseases such as SLE,intestinal lymphangiectasia. Treatment should be focused on primary disease.

Objective To investigate correlation between microRNA (miR-494) and TH 17 cell differentiation in murine cervical heterotopic cardiac transplant model.Method The heterotopic cardiac transplant models of Balb/c→C57BL/6 mice were established as experimental group,and those of C57BL/6→C57BL/6 mice as control group.Real time-polymerase chain reaction(PCR) was used to detect miR-494 and interleukin(IL)-17A mRNA expression in the grafts.CD4+ T cells,CD8+ T cells and CD45+ myeloid cells were isolated from the grafts,and miR-494 and IL-17 mRNA expression was detected.In vitro,lymphocytes in the spleen from C57BL/6 mice were harvested,and CD4+ T cells were isolated with MACS and then stimulated to TH 1,TH 2,TH 17,Treg subset cells.The expression of IL-17A mRNA and miR-494 in different T subsets was examined by Reverse transcription-polymerase chain reaction(RT-PCR).Result Two grafts from each study group were harvested on the 7th day post-transplantation.In experimental group,the IL-17A mRNA expression was increased,while the expression of miR-494 was decreased as compared with control group with the difference being significant between two groups.The expression of IL-17A rnRNA in CD4+ T cells of the grafts was significantly increased,while that expression of miR-494 was decreased.In vitro,the expression of miR-494 in TH 17 cells was significantly lower than that in TH 1,TH 2 and Treg cells.Conclusion miR-494 is related closely to TH 17 cells differentiation in the transplant rejection,which may play a role in transplant rejection through regulating TH 17 cells.

Objective: To study the relationship between vitamin A (VA) supplementation and the level of IgG antibody to measles strengthened vaccination. Method: Fifty-three school children aged 5-13 years were selected as the test group of VA supplementation, and fifty-two school children as the control group whose ages and sex matched with the test group. The test group was supplicd po the VA pills (2 500IU) at the same time with measles strengthened vaccination for one month. The level of serum VA was analyzed by HPLC. Measles antibody IgG was detected by ELISA. Results: One month after VA supplementation, sernm VA in test group was 376.5?74.2 ?g/L, showing statistical increase over before. The positive rates of measles antibody in the test group were increased from 69.8% to 100%, and the protective rate from 5.6% to 60.4%. The positive rates of measles antibody in the control group were increased from 71.2% to 100%, and the protective rates from 0% to 17.4%. The protective rate in the test group was statistically higher than the control. Conclusion: Simultaneous VA supplementation (especially for the children of VA deficiency) and measles strengthened vaccination contribute to the increase of measles antibody IgG in school children.