Objective To evaluate hepatic functional reserve and investigate the clinical value through measuring hepatic functional blood flow by D-sorbitol clearance rate and liver volume changes with CT. Methods Ninety-two patients with portal hypertension due to posthepatic cirrhosis were investigated (cirrhosis group). Twenty healthy volunteers were used as control group. D-sorbitol was infused intravenously at a steady rate. Blood and urine were collected and recorded once before infusion and at 120, 150 and 180 min after infusion, and their concentrations of D-sorbitol were examined by enzyme spectrophotometry. From pharmacokinetic equations, hepatic clearance rate of D-sorbitol (CLH) was calculated. Total hepatic blood flow (QTOTAL) was measured by Doppler sonography, intrahepatic shunt rate (RINS) was obtained. The liver volume change rate was obtained in patients with cirrhosis through the abdominal CT scan. The relations among the indicators, Child classification and postoperative complications were studied. Results After D-sorbitol was infused intravenously for 120 min, the plasma concentration was at the steady state. The plasma concentration was (0.189±0.05) mmol/L in control group and (0.358±0.06) mmol/L in cirrhosis group (Plt;0.01). CLH was (1 248.3±210.5) ml/min in control group and (812.7±112.4) ml/min in cirrhosis group (Plt;0.01). Although QTOTAL in cirrhosis group was declined, compared with the control group 〔(1 280.6±131.4) ml/min vs. (1 362.4±126.9) ml/min〕, Pgt;0.05, while RINS increased markedly 〔(36.54±10.65)% vs. (8.37±3.32)%, Plt;0.01〕. In cirrhosis group, the mean liver volume of Child A, B and C patients were (1 057±249) cm3, (851±148) cm3 and (663±77) cm3 respectively. There were significant differences among the mean liver volume (Plt;0.05). The liver volume was significantly smaller in Child B and C patients than that in Child A (Plt;0.05, Plt;0.01). When CLH was less than 600 ml/min, and liver volume decreased by more than 40%, postoperative complications increased significantly. CLH and the liver volume change rate were not in absolutely good accordance with Child classification. Conclusion The hepatic clearance of D-sorbitol and the quantitative determination of the liver volume with CT can be an objective evaluation of the liver metabolism of the inherent capacity and the hepatic functional blood flow changes. It contributes to the correct understanding of the hepatic functional reserve and lay the foundation for determining a reasonable treatment plan, surgical methods and time.
【Abstract】 Objective To explore the optimal dosage, timing and cytotoxicity of bromodeoxyuridine (BrdU) labelling for rabbit adipose-derived stromal stem cells (ADSCs) in vitro so as to confirm its feasibil ity for stem cells labell ing and tracer means. Methods Six rabbits were used in this experiment, aged 8-12 weeks, weighing 1.5-2.0 kg and neglecting their gender. 1-2 mL fat was removed, the ADSCs were isolated and cultured using the adherence method in vitro . The 3rd passage of ADSCs was incubated with BrdU at 5, 10, 15 and 20 μg/mL (groups A, B, C and D）for 12, 24, 48 and 72 hours to identify the optimal BrdU concentration and incubating time for cell labell ing. Immunohistochemistry and trypanblau strain were performed respectively to calculate the labell ing index (positive rate) and the cells’ activity for different time after BrdU labell ing. The ADSCs without BrdU labell ing were used as control (Group E）. Results The main appearance of primary ADSCs was short fusiform shape, and of the 3rd passage ADSCs long fusiform shape. The 3rd passage of ADSCs could differentiate into osteoblastsand adipocytes under corresponding inductive medium. The ADSCs’ nucleus show green fluor under fluorescence microscope after labeled by the BrdU. The labell ing ratio increased in groups A, B, C and D after incubating 12 hours, the mean labell ing ratio were 30.6％ ±2.3％，32.4％ ±1.9％，45.8％ ±1.8％，50.8％ ±3.1％ , respectively, and the labell ing ratio of Group E was 0. There were significant differences between groups C, D and Group A (P lt; 0.01). The labell ing ratio of groups A, B, C and D were 45.9％ ±2.0％，87.9％ ±3.3％，90.6％ ±2.9％，91.7％ ±3.2％，respectively after 24 hours and the labell ing ratio of Group E was 0. There were significant differences between groups B, C, D and Group A (P lt; 0.01). The results of all groups after incubating48 hours and 72 h ours were similar to that after incubating 24 hours. The cell counting of groups A, B, C and D were better than that of Group E, but showing no siginificant differences（P gt; 0.05）. Conclusion The most appropriate time for BrdU labell ing ADSCs is 48 hours, the most appropriate concentration is 10 μg/mL. The labell ing rate is high and cytotoxicity is l ittle.
Objective To investigate the method and effectiveness of wide local excision combined with Mohs micrographic surgery for dermatofibrosarcoma protuberans (DFSP). Methods Between January 2007 and January 2010, 17 patients with DFSP were treated. There were 9 males and 8 females with an average age of 33.2 years (range, 16-55 years). Thelesions were located at head and neck (2 cases), trunk (12 cases), extremity (2 cases), and perineal region (1 case). There were 6 cases of primary DFSP and 11 cases of relapsed DFSP. The lesions presented as single or multitude nodules or fusion nodules with skin withering, scar, en plaque in the center and with ill-defined margins. The diameter of lesions ranged from 0.8 to 9.7 cm (mean, 4.3 cm). No distant metastasis or lymphatic metastasis occurred in all cases. After tumors resection by wide local excision combined with Mohs micrographic surgery, the wounds were repaired by direct suture in 3 patients, skin grafting in 9 patients, and local skin flap in 5 patients. Results Wide local excision and Mohs micrographic surgery were carried out once in 13 patients, twice in 3 patients, and three times in 1 patient with an average operation time of 98.6 minutes (range, 56-219 minutes). Primary heal ing of wound and donor site were achieved with no necrosis of skin grafting and skin flap. All patients were followed up 8-34 months (mean, 21.7 months) with no recurrence. Conclusion Wide local excision combined with Mohs micrographic surgery could treat DFSP, which has the advantages of shorter operation time, radical resection, and less injury.
As most patients of central serous retinopathy (CSC), the symptoms of acute onset will alleviate by oneself after 4-6 months. About 30%-50% of patients with CSC experience chronic or recurrent cases. Resulting in persistent neurosensory detachments and subretinal fluid, causing significant vision loss. Mineralocorticoid receptor (MR) is a kind of nuclear hormone receptors, plays a role in theregulation of water and electrolyte balance. Excessive MR signaling is associated with many diseases. Study found that MR antagonists decreased the thickness of the retina and improved in vision, there was no serious adverse reactions during the period of treatment for chronic CSC. Initial dose of MR antagonists was 25 mg per day, 1 week later, dosage was increased to 50 mg per day, and treatment for about 3 months. There is no conclusive effective treatment and the dosage are still unknown. MR antagonists may be a safe and effective way to treat chronic CSC, though evidence is scant. Prospective, multicenter, large-scale trials is required.
Objective To investigate the cell growth inhibition and apoptosis induced by rapamycin on human hepatocellular carcinoma Bel-7402 cells and to study the role of mitochondrium membrane potential in the process of apoptosis. Methods Bel-7402 cells in vitro were given 5, 10, 20, 30, 40 and 50 nmol/L different concentrations of rapamycin, and the cell growth inhibiting ratio of Bel-7402 was assessed by MTT assay. The changes of morphology of Bel-7402 were observed by Hoechst 33258 staining and flow cytometry (FCM), respectively; The cell mitochondrial membrane potential was detected by using JC-1 staining method. Results Rapamycin could inhibit the growth of Bel-7402 cells significantly by inducing apoptosis, and the growth suppression and the cell apoptosis both presented time-effect relationship and were also dose-dependent. The rates of inhibiting and cell apoptosis after 72 h exposure to 50 nmol/L rapamycin were significantly higher that those of other groups (P＜0.01). Typical morphological changes of cell apoptosis were observed very clearly after the Bel-7402 cells had been exposed to rapamycin for 48 hours using Hoechst 33258 staining method, and it was also observed that the mitochondrial membrane potential decreased when apoptosis occured (P＜0.01). Conclusion Rapamycin could inhibit the growth of Bel-7402 cells by inducing cell apoptosis, and the descent of mitochondrial membrane potential may play an important role in the process of cell apoptosis.
Objective To construct the expression vector of HLA-G-shRNA and investigate the effect of HLA-GshRNA from NK cell lysis. Methods Four HLA-G shRNA plasmids were constructed and transiently transfected to Bel-7402 cell lines， the levels of mRNA and protein of HLA-G were detected by Real-Time PCR and Western blot. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay. Results The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one which we designed, and no aberrations such as mutation，deletion or insertion occurred. The expressions of HLA-G confirmed by Real Time-PCR and Western blot were significantly down-regulated. Bel-7402 cell lines transfected HLA-G shRNA showed higher lytic activity （P＜0.01）. After KIR2DL4 receptor blocked，lytic activity of NK-92 MI cell were decreased （P＜0.01）. Conclusions HLA-G shRNA plasmids are successfully constructed and HLA-G down-regulated can increase NK cytolysis against Bel-7402 cell. After HLA-G combines with KIR2DL4 receptor at the surface of NK cells， the inhibition effect is transferred.
Objective To investigate the expression of human leukocyte antigen-G （HLA-G） in hepatocellular carcinoma （HCC） tissues， and to evaluate the prognosis of patients after liver transplantation. Methods The clinical data of 83 patients with HCC who underwent liver transplantation from January 2004 to May 2008 in the Liver Transplantation Center of the Third Affiliated Hospital of Sun Yat-sen University were analyzed retrospectively. The expression of HLA-G in HCC tissues was detected by using immunohistochemical analysis. The Kaplan-Meier method was used to evaluate the cumulative survival rate and tumor-free survival rate. Log-rank test and Cox regression model were used to analyze the single and muti-factor for tumor-free survival rate, respectively. Results Among the 83 patients，there were tumor recurrence in 35 patients （42.2%）. The 1-year，3-year, and 5-year of cumulative survival rate was 97.2%，89.8%, and 43.1%, respectively. The 1-year，3-year， and 5-year of tumor-free survival rate was 93.6%，68.9%, and 38.7%, respectively. The positive rate （68.7%） of HLA-G expression in HCC tissues was significantly higher than that in paracancerous tissues （15.7%）, P＜0.01. A significant association was found between expression of HLA-G and tumor size， vascular invasion, and pathology differentiation （P＜0.05）. Single factor analysis showed that the expression of HLA-G （P＜0.01）， tumor size （P＜0.05）， vascular invasion （P＜0.01）， and pathology differentiation （P＜0.01） effected on tumor-free survival rates of HCC patients after liver transplantation. The tumor-free survival rate in positive expression of HLA-G group was significantly lower than that in negative expression of HLA-G group （P＜0.01）. Cox regression model analysis showed that the expression of HLA-G （P＜0.05）， vascular invasion （P＜0.01）， and pathology differentiation （P＜0.05） were independent risk factors that affected the tumor-free survival rate of HCC patients after liver transplantation. Conclusions There is expression of HLA-G in HCC tissues. The independent risk factors that affecting the tumor-free survival rate of HCC patients after liver transplantation include the expression of HLA-G， vascular invasion, and pathological differentiation. Taking interferential measures for patients with positive expression of HLA-G and strict selection of indication of liver transplantation for HCC can reduce the recurrence rate of tumor.