BACKGROUND:PA6 cells are bone marrow stromal stem cells from the mouse skul , and scientists have found that PA6 cells co-cultured with some kinds of stem cells have shown neural differentiation, based on which, PA6 cells can be used for repair of nerve injury. Therefore, researchers give more and more attentions to PA6, but few studies have addressed isolation and culture methods of bone marrow stromal stem cells from the skul . OBJECTIVE:To isolate and culture bone marrow stromal stem cells from the skul of Sprague-Dawley rats and to observe cellular morpholopy in vitro and perform immunofluorescence identification. METHODS:Under sterile conditions, newborn Sprague-Dawley rat’s skul was cut into pieces. Smal skul pieces were washed using Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum. Single cellsuspension was made, and placed in culture flask. The culture medium was changed many times for cellpurification. The second passage of cells was obtained for morphology observation under an inverted microscope. cellsurface markers were detected by using immunofluorescence staining. RESULTS AND CONCLUSION:After the primary culture for 24 hours, cells exhibited adherent growth;after 3 days, cells were increased in number, and presented with irregular shapes, such as polygon, triangle, spindle and flat shape. After passage, cells were uniform in morphology, arranged in radial and bunch patterns, and exhibited strong adherent ability. Some cells grew in cluster, and proliferated faster than primary cells. Immunofluorescence staining showed that cells were positive for CD105, CD73, CD44, CD90, but negative for CD45, D34,CD14, HLA-DR. Results indicate that this method can obtain bone marrow stromal stem cells.

Objective To evaluate the diagnostic accuracy of ultrasonography (US) and CT for thyroid masses. Methods Seventy-one patients with thyroid masses (13 with malignant and 58 with benign tumors) confirmed by operation and pathology were collected. The apprearances of CT and US before operation were analyzed. The apperanecs of CT and US, including the clean edge of masses, calcification and cystic degeneration necrosis were compared with those of pathologic findings. Results The numbers, cystic changes, configuration, verge, calcify and enlarge cervical lymph nodes of thyroid lesions had statistical difference in CT and US (P＜0.05). There was also difference in the internal echo, ring of halo on US and the sign of halo on plane CT (P＜0.05). The edge of thyroid mass could be displayed more clearly with US than CT (P＜0.05), however, it was similar with CT in the display of calcify and cystic changes (P＜0.05). Conclusion Both CT and US can display thyroid mass clearly. Combing of CT and US could improve the accuracy rate of diagnosis.

Objective To summarize the clinical experience of the application of bortezomib desensitization regime prior to secondary renal transplantation in a highly-sensitized recipient. Methods At 13, 10 and 6 d prior to secondary renal transplantation, one patient positive for donor specific antibody (DSA) was subcutaneously administered with bortezomib at a dose of 1.3 mg/m2 combined with a low dose of immunoglobulin. Postoperatively, immunosuppressive regime of tacrolimus (FK506), mycophenolat sodium and methylprednisolone was adopted. The serum creatinine (Scr), blood urea nitrogen (BUN) levels, FK506 concentration, DSA titre, C3d binding DSA (C3d-DSA) titre, pathological biopsy of the renal graft and adverse reactions were observed. Results During 12-month follow-up after administration of bortezomib, the Scr level was declined and maintained at 130 μmol/L, and the BUN level was remained at 3.9 mmol/L. The DSA level was significantly decreased and the C3d-DSA was negative. At postoperative 4 and 9 months, pathological biopsy of the renal graft revealed that the patient was positive for C4d, prompting the chronic active antibody mediated rejection (AMR). The patient presented with grade Ⅲ peripheral neuropathy. Conclusions Application of preoperative bortezomib desensitization regime can effectively down-regulate the DSA level in the recipient and avert the incidence of acute rejection in highly-sensitized patients undergoing secondary renal transplantation. Comprehensive treatment using bortezomib is recommended for preoperative desensitization in the highly-sensitized transplant recipients.

Objective:To perform a prospective cohort study to identify individual susceptibility of glucocorticoid (GC) -associated osteonecrosis of the femoral head (GA-ONFH) and their clinical and genetic risk factors. Methods:The present prospective cohort study enrolled patients who received their first GC therapy between July 2015 and January 2018 at Zhongshan Hospital. All patients did not receive any GC treatment before enrollment. Further, they planned to start GC treatment with the dose (equivalent prednisone) of ≥30 mg/d, lasted ≥3 weeks, or pulse dose ≥200 mg/d, lasted ≥3 d. Blood samples were collected before GC treatment to evaluate bone metabolism and its released factors. Hip MRI was performed at the 1st, 3rd, 6th, 12th and 24th month to diagnose GA-ONFH. All patients were followed-up for ≥2 years. The endpoint was regarded as diagnosis of GA-ONFH or completion of 2 years follow-up. Lasso regression was performed to determine which clinical features were associated with GA-ONFH. A nested case-control sub-cohort (A, n=12) was established prospectively based on the main cohort by 1∶1 matching. Whole exome sequencing was performed to screen differential and functional candidate single nucleotide polymorphisms and insertion-deletions (SNP/InDels). Another sub-cohort (B, n=50) was constructed retrospectively in patients with GA-ONFH and non-ONFH patients received standard high dose GC treatment for more than two years. The candidate SNP/InDels were verified by Sanger sequencing based on the patients from sub-cohort B. Results:A total of 96 patients were enrolled of which 88 of them (32 males and 56 females, mean age 42.30 years) completed follow-up. Eight cases (9.1%) were diagnosed with GA-ONFH. The median time from the start of GC therapy to the diagnosis of ONFH was 53.00(34.00,13.50) days. The baseline characteristics, such as age, sex and body mass index, indicated no significant difference between the ONFH group and the non-ONFH group. The cumulative GC dose of the ONFH patients in the first month was higher than that of non-ONFH [32.74(29.55, 47.05) mg/kg vs. 24.00(21.10, 29.45) mg/kg, Z=-2.410, P=0.016]. However, there was no significant difference of patients who underwent pulse therapy (37.5% vs. 10.0%, adjusted χ 2=2.829, P=0.093). The ratio of serum apolipoprotein B/apolipoprotein A1 (ApoB/ApoA1) in patients with ONFH was higher than that in non-ONFH group before GC use [0.95(0.80, 1.50) vs. 0.70(0.60, 0.80), Z=-2.875, P=0.000]. Due to the multicollinearity, Lasso regression model was performed to reduce overfitting. All variables were included in the model. The results suggested that higher ApoB/ApoA1 ratio, lower serum β-c-terminal telopeptide (β-CTX) and higher cumulative GC dose in the first month were the top three risk factors of GA-ONFH. This model had an accuracy of 0.982 in internal validation. Seven differential candidate SNP/InDels were found by whole exome sequencing of sub-cohort A. We further verified these SNP/InDels in sub-cohort B. The patients with COLEC12 mutation (rs2305027, G1816A) were at risk of GA-ONFH ( OR=6.00, 95% CI: 1.17, 30.73). Conclusion:Higher first-month GC dose, lower serum β-CTX level before treatment, higher ApoB/ApoA1 ratio and COLEC12 mutation (rs2305027, G1816A) could increase the risk of GA-ONFH.

Objective:To explore the relationship between new biomarkers in donor blood and delayed graft function after kidney transplantation and evaluate the clinical value of new biomarkers in the diagnosis of DGF (delayed graft function).Methods:For recipients of donor kidney transplantation from August 2016 to December 2017, blood samples were collected from operations of donor organ resection within 12 hours of the day. Enzyme-linked immunosorbent assay (ELISA) was employed for detecting neutrophil gelatinase-associated lipocalin (NGAL), L-type fatty acid binding protein (L-FABP), kidney injury molecule-1 (KIM-1) and interleukin-18(IL-18). They were divided into DGF and EGF (early graft function) groups according to the diagnosis of DGF. The inter-group differences of four new biomarkers were calculated. Receiver operating characteristic curve (ROC curve) was plotted for finding the best positive cutoff value and the sensitivity and specificity of new donor blood marker for diagnosing delayed graft function were calculated.Results:Among them, 8 had postoperative DGF and 62 had none. The overall incidence of DGF was 11.43%. The serum concentration of NGAL was 521.01±132.84 ng/ml in DGF group versus (299.99±100.03) ng/ml in EGF group ( P<0.001). The ROC curve was plotted. With a NGAL concentration >425.15 ng/ml, the sensitivity and specificity for diagnosing DGF were 87.5% and 90.3% respectively. The area under the curve (AUC) was 0.891. The serum concentration of IL-18 was (14.10±12.36) ng/ml in DGF group and (4.61±1.83) ng/ml in EGF group ( P=0.047). With a IL-18 concentration of >5.345 ng/ml, the sensitivity and specificity for diagnosing DGF were 100% and 64.5% respectively. The AUC was 0.914. No significant inter-group difference existed in serum L-FABP/KIM-1. The sensitivity of donor creatinine in the diagnosis of DGF was 62.5%, specificity 75.8% and AUC 0.692. Conclusions:With an excellent level of sensitivity and specificity, an elevated concentration of NGAL/IL-18 in donor blood is superior to traditional creatinine in the diagnosis of DGF after renal transplantation.