BACKGROUND:It is accepted that the best method for spermatogonial stem cells separation is using artificial cryptorchism model combined with surface makers.OBJECTIVE:To explore the feasibility of separation spermatogonial stem cells with ?6-integrin and c-kit as specific surface markers.DESIGN,TIME AND SETTING:The randomized control experiment was performed at the Renmin Hospital of Wuhan University from May to December 2006.MATERIALS:Forty adult,white Kunming mice with 6 weeks old were randomly divided into cryptorchidism and control groups,with 20 animals in each group.METHODS:Artificial cryptorchidism model was prepared by made an incision at the median of abdomen,and testis was pulled into abdominal cavity,which was fixed at the each side of lateral abdominal wall.There was no treatment in the control group.The single cell suspension of seminiferous epithelium was obtained by traditional two step enzyme digestion at 2-3 months after operation.FITC-conjugated anti-?6-intergrin antibody and PE-conjugated anti-c-kit antibodies were added.Then the cells with low side scatter light-scattering properties were sorted and positively stained for ?6-intergrin and negative c-kit expression.Meanwhile,the viability of the isolated cells was assessed by trypan blue staining.MAIN OUTCOME MEASURES:The morphological changes of cryptorchidism,and the sorting results of spermatogonial stem cells.RESULTS:Cell distribution in seminiferous tubule was disorder with reduced numbers.The layer and lumens were disappeared,and cell division phase could be seen in the center of tubules.Compared to the control group,the testicular cells in the cryptorchidism group were increased in the side scatterlow,Forward scatterhi areas,with figure left-upward displacement.The distribution of ?6-integrin+ and c-kit cells were deviated each other,it named that most ?6-integrin+ cell were not spermatogonial stem cells,so do the c-kit-cells.Only 2.8% of testicular cells exhibited side scatterlow,?6-integrin+,and c-kit-,which were spermatogonial stem cells in the cryptorchidism group.And trypan blue staining showed that over 95% of them were viable.CONCLUSION:Using the two surface markers to sort spermatogonial stem cells can advance the purity of the spermatogonial stem cells in cell suspension,but the specificity is insufficient.

Objective To investigate the clinical features , diagnosis and key technique points of laparoscopic partial adrenalectomy for small adrenal pheochromocytoma . Methods From Oct.2006 to Jun. 2011, clinical data of 32 cases with small adrenal pheochromocytoma (≤3.0 cm) were collected and retro-spectively analyzed .Hypertension was observed in 12 patients, whereas 20 patients presented with adrenal incidentaloma .Thirteen patients had a left adrenal neoplasm , eighteen patients had a right adrenal tumor , while one patients had bilateral tumors .The positive rate of plasma-free metanephrines ( MNs) and 24-hours urinary catecholamine (CA) in diagnosing small renal pheochromocytomas was 92.6%(25/27) and 81.3%(26/32) respectively.The main localization diagnosis included ultrasonography , 131I-MIBG, and CT or MRI, with positive rates of 71.9%(23/32), 93.8%(15/16) and 96.9%(31/32) respectively.All the laparoscopic adrenalectomies were performed retroperitoneally .During the surgery , the internal part of the adrenal gland closing to the retroperitoneum was dissected first , and the whole adrenal gland was resected completely. Results Partial adrenalectomy was performed for 30 cases and radical adrenalectomy for 2 ca-ses.All operations were successful without perioperative or postoperative complications .The maximum diame-ter of tumor was 1.7±0.2 (1.0-3.0) cm.Histopathological results showed that all the cases were benign pheochromocytoma.The operative time was 82 (40-210) min.The estimated blood loss was 57 (20-180) ml.No patient required blood transfusion . Conclusions Plasma-free MNs, 24-hours urinary CA and VMA are important qualitative examinations in detection of adrenal pheochromocytoma .Ultrasonography , CT,MRI, and 131 I-MIBG are important in the localization of adrenal tumors .Retroperitoneal laparascopic partial adrenalectomy is the preferred choice in the management of small adrenal pheochromocytoma .Dissecting the internal part of the adrenal gland closing to the retroperitoneum first and exploring the whole adrenal tissue are the key technique points during the operations .

Aim To synthesize and identify artificial antigen of podophyllotoxin for the production of podophyllotoxin polyclonal antibody. Methods The hapten was synthesized by two different chemical approaches and characterized by TLC, IR, NMR, and MS. Mixed anhydride reaction (MAR) and active ester method (AEM) were used to couple the podophyllotoxin to carrier proteins (BSA and OVA). Characterization of artificial antigens was done by using spectroscopy and electrophoresis. The anti-podophyllotoxin polyclonal antibodies were obtained through immunizing rabbits. Results The results from IR, NMR and MS showed that 4-O-succinoyl podophyllotoxin (hapten) was successfully synthesized. The coupling molar ratios of the hapten and carrier proteins were 88.6 for Hapten-BSA1, 40.3 for Hapten-BSA2, 17.8 for Hapten-OVA1, and 54.2 for Hapten-OVA2. Hapten conjugates coupled with BSA yielded two sets of the specific and affinitive polyclonal antibodies. One set of antibodies showed an IC50 value of 2.21 μg·mL -1 with a detection limit of 0.12 μg·mL -1. Conclusion Antigenic conjugates were artificially synthesized, and based on these artificial antigens, polyclonal antibodies against podophyllotoxin were raised from rabbits immunized with two different immunogens and characterized with an indirect ELISA format.

Objective To investigate the effect of lysimachia capillipes(LC) on proliferation of human pancreatic cancer cell line BxPC-3 in vitro and explore the potential mechanism.Methods BxPC-3 cells were treated by LC in different concentrations of 2,4,8,16,32,64 μg/ml for 48 and 72 hours,respectively,using untreated cells as control.The survival rate of BxPC-3 cells was measured by MTT method.The half inhibition concentration (IC50) of LC was determined by drawing growth curve.BxPC-3 cells were treated by LC in the concentration of IC50 (LC group),and cell apoptosis and cell cycle were examined by using flow cytometry.The protein expression of PARP and capase-3 was detected by Western blotting.Results LC in the concentration of 8-32 μg/ml inhibited the survival rate of BxPC-3 cells in a dose-dependent manner.After exposure to 15 μg/ml LC for 48 h,the apoptosis rate of BxPC-3 cells was increased [(17.3 ± 0.31)％ vs (1.5 ± 0.22) ％],but the cell cycle was not affected.The expression of caspase-3 protein was up-regulated [(2207.2 ± 92.0) vs 149.1 ± 10.2] and PARP protein was down-regulated [(36.1 ± 4.8) vs 1593.4 ±29.7] than control group,and the differences were statistically different (all P value ＜ 0.05).Conclusions LC can inhibit the growth of BxPC-3 cells,and the potential mechanism was associated with the induction of cell apoptosis by LC via upregulating caspase-3 protein expression and decompsing PARP protein.

Objective To investigate the diagnosis and treatment of extra-adrenal pheochromocytoma(EAP). Methods The clinicsl data of 37 cases of EAP from April 2003 to April 2010 were retrospectively analyzed.Hypertension was observed in 31 cases.The typical triad of headache,palpitation and sweating was observed in 12 cases.The positive rate of plasma-free MNs and 24-hour urinary CA in diagnosing EAP was 96.8%(30/31) and 86.5% (32/37) respectively.The main localization diagnosis included ultrasonography,CT,MRI and 131I-MIBG,with positive rates of 91.7% (33/36),97.0%(32/33),90%(9/10) and 82.6%(19/23) respectively.Two patients underwent radiotherapy, and the remaining 35 cases underwent surgical treatment. Results Among the total of 37 cases,32 cases were single tumor,and five were multiple tumors.The anatomic locations of the single tumors were as follows: 14 wre adjacent to the abdominal aorta,seven in the bladder,four adjacent to the inferior vena cava,four adjacent to the renal hilum,two adjacent to the lilac blood vessel and one in the upper pole of the right kidney.Thirty cases underwent complete tumor resection,three cases underwent tumor resection plus right nephrectomy and two cases underwent partial cystectomy.Twenty-four cases were diagnosed benign and 11 cases were diagnosed malignant by pathological examination.Among 31 cases with preoperative hypertension,postoperative blood pressure returned to normal in 23 patients,blood pressure descended mildly in six cases and blood pressure was still hypertensive in two cases.Thirty-four patients were followed up for five months to seven years,during which five cases had tumor recurrence or metastases and five cases died postoperatively. Conclusions EAP is a rare neuroendocrine tumor and its accurate diagnosis is rather difficult.Plasma-free MNs and 24-hour urinary CA are important qualitative examinations.Ultrasonography,CT,MRI and 131I-MIBG scintigraphy are important methods for the localization of the tumor.Transperitoneal resection of the tumor is the preferred choice of management and adequate perioperative preparation is the key to a successful operation,including bringing down blood pressure,expanding blood volume and correcting arrhythmia.Patients with malignant EAP may be treated with 131I-MIBG after surgical therapy.

Objective To explore the clinical efficacy and safety of off-clamping in robotic-assisted partial nephrectomy (RAPN) for the treatment of renal tumors.Methods From January 2015 to March 2017,the data of 48 patients who underwent off-clamping RAPN were reviewed retrospectively.There were 31 males and 17 females,and the mean age was 57 years (range:23-84 years).The mean tumor size was 3.1 cm (range:1.2-6.4 cm),with the upper,middle,and lower polar tumors account for 35.4％,27.1％,and 37.5％,respectively.The clinical tumor stage was T1N0M0 in all 48 cases,according to the AJCC tumor staging system for renal cancer.Results RAPNs were performed successfully in all 48 cases,without conversion to open surgery.In those patients,the application of off-clamping in robotic-assisted partial nephrectomy was performed in 44 cases.The renal artery and vein was exposed,dissected,isolated and then clamped in 4 cases due to bleeding.The mean surgical time was 85 min (range:75-185 min).The mean estimated blood loss was 134 ml (range:60-270 ml),and no blood transfusion was needed.The wound surface was closed using interrupted suture with Hem-o-lok clips securing each needle point.The mean time for renorrhaphy was 22 min (rang:11-31 min).No intraoperative severe complications such as vascular injury,trauma of abdominal organ occurred.There were 5 complications,including 2 cases of hematuria,2 cases of delayed healing of incision,and 1 case of pneumohypoderma.The pathological diagnosis included 40 cases of renal clear cell carcinoma,3 cases of papillary renal cell carcinoma,and 5 cases of angiomyolipomas.No tumor recurrence or distant metastasis was observed during the average follow-up of 17 months (range 3-27 months).Conclusions Off-clamping RAPN is safe and feasible approach to excise certain kidney tumors.It carries the benefits of less complication,quick recovery,and less ischemia reperfusion renal injury.Off-clamping RAPN would be suitable for those patients with solitary kidneys,renal insufficiency,and bilateral tumors.

Background: Cardiopulmonary resuscitation (CPR) strategies in COVID-19 patients differ from those in patients suffering from cardiogenic cardiac arrest. During CPR, both healthcare and non-healthcare workers who provide resuscitation are at risk of infection. The Working Group for Expert Consensus on Prevention and Cardiopulmonary Resuscitation for Cardiac Arrest in COVID-19 has developed this Chinese Expert Consensus to guide clinical practice of CPR in COVID-19 patients. Main recommendations: 1) A medical team should be assigned to evaluate severe and critical COVID-19 for early monitoring of cardiac-arrest warning signs. 2) Psychological counseling and treatment are highly recommended, since sympathetic and vagal abnormalities induced by psychological stress from the COVID-19 pandemic can induce cardiac arrest. 3) Healthcare workers should wear personal protective equipment (PPE). 4) Mouth-to-mouth ventilation should be avoided on patients suspected of having or diagnosed with COVID-19. 5) Hands-only chest compression and mechanical chest compression are recommended. 6) Tracheal-intubation procedures should be optimized and tracheal-intubation strategies should be implemented early. 7) CPR should be provided for 20-30 min. 8) Various factors should be taken into consideration such as the interests of patients and family members, ethics, transmission risks, and laws and regulations governing infectious disease control. Changes in management: The following changes or modifications to CPR strategy in COVID-19 patients are proposed: 1) Healthcare workers should wear PPE. 2) Hands-only chest compression and mechanical chest compression can be implemented to reduce or avoid the spread of viruses by aerosols. 3) Both the benefits to patients and the risk of infection should be considered. 4) Hhealthcare workers should be fully aware of and trained in CPR strategies and procedures specifically for patients with COVID-19.