Objective To establish a new method based on Taqman's fluorescent ARMS-PCR technique for detecting the KRAS mutations in human paraffin embedding tissue samples,which is a reference value of clinical medication.Methods Specific primers and probes were designed based on the conserved sequences of KRAS and internal reference genes(GAPDH).Taqman's fluorescent ARMS-PCR detection systems and sequencing systems were established for KRAS detection.In this paper,paraffin embedded tissue samples of 200 patients with colorectal cancer(CRC)are detected and compared with gold standard Sanger sequencing.The results of KRAS gene mutation are statistically analyzed,and the consistency of the two detection methods is analyzed.Results This method is able to detect KRAS gene mutations in human paraffin embedding tissue samples.And the results are 100％consistent with Sanger sequencing.Conclusion The method based on Taqman's fluorescent ARMS-PCR established in this research can detect KRAS mutations simply,quickly and accurately,which is valuable for clinical medication and suitable for clinical application.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

Objective To evaluate the effect of microvascular invasion (MVI) on prognosis of recipients after liver transplantation for primary liver cancer (liver cancer). Methods Clinical data of 177 recipients after liver transplantation for liver cancer were retrospectively analyzed. All patients were divided into the MVI-positive group (n=64) and MVI-negative group (n=113) according to postoperative pathological examination results. Clinical data were statistically compared of all recipients between the negative and positive MVI groups. The prognosis and risk factors of liver transplantation recipients for liver cancer were analyzed. Results Among 177 recipients, 64 cases (36.2%) were positive for MVI and 113 (63.8%) negative for MVI. Compared with the MVI-negative recipients, MVI-positive recipients had significantly lower degree of tumor differentiation, higher preoperative alpha-fetaprotein (AFP) level, larger maximal tumor diameter, a larger quantity of tumors, more satellite lesions and more recipients who did not meet the Milan criteria (all P < 0.05). The 1-, 3- and 5-year overall survival (OS) and recurrence-free survival (RFS) of recipients after liver transplantation for liver cancer were 80.2%, 62.1%, 58.5% and 66.3%, 57.5%, 51.2%, respectively. The 1-, 3- and 5-year OS and RFS of MVI-positive recipients were 70%, 39%, 35% and 53%, 39%, 33%, significantly lower than 86%, 75%, 72% and 73%, 68%, 63% of their counterparts negative for MVI (all P < 0.05). Cox regression analysis showed that the maximal tumor diameter >8 cm, preoperative AFP level ≥20 ng/mL, low degree of tumor differentiation and positive MVI were the independent risk factors for OS of recipients after liver transplantation for liver cancer (all P < 0.05). Positive MVI, low degree of tumor differentiation and preoperative down-staging failure were the independent risk factors for RFS of recipients after liver transplantation for liver cancer (all P < 0.05). Conclusions MVI is of significant clinical value in predicting clinical prognosis of recipients after liver transplantation for liver cancer.

Objective To evaluate the effect of the different Child-Pugh classification on the recurrence and survival of hepatocellular carcinoma (HCC) recipients after liver transplantation. Methods Clinical data of 125 HCC recipients undergoing liver transplantation were retrospectively analyzed. The 3-year disease-free survival (DFS) and overall survival (OS) rates were calculated by Kaplan-Meier survival curve. The independent risk factors probably affecting the recurrence and survival of HCC recipients after liver transplantation were identified by using Cox's proportional hazards regression model. Results The median follow-up time was 25.6 months. The 3-year DFS and OS rates were 68.4% and 65.7% for all patients. The 3-year DFS and OS rates in 113 patients with Child-Pugh class A/B HCC were 68.6% and 66.2%, whereas 66.7% and 65.6% for 12 patients with Child-Pugh class C HCC with no statistical significance (all P>0.05). Cox's proportional hazards regression model demonstrated that vascular invasion (P=0.001)and the number of tumors>3 (P=0.025) were the independent risk factors for the postoperative recurrence of HCC in recipients undergoing liver transplantation. Alpha fetoprotein (AFP)>400μg/L (P=0.035), vascular invasion (P=0.031) and number of tumors>3 (P=0.008) were the independent risk factors affecting the survival of HCC patients. Conclusions The postoperative prognosis does not significantly differ between Child-Pugh class C and A/B HCC patients after liver transplantation. AFP, vascular invasion and number of tumors are the risk factors affecting the clinical prognosis of HCC patients after liver transplantation. Liver transplantation is an efficacious treatment for HCC patients with Child-Pugh class C.

Objective To explore the features of peripheral blood immune cells in long-term survival recipients after liver transplantation.Methods The expression of T subsets (Th1,Th2,Th17,Th22,Tregs),NK cells,NKt cells,Bregs,MDSC in long-term survival recipients (postoperative follow-up time ≥5 years,30 cases),short-term survival recipients(postoperative follow-up time ≤1 year,15 cases) and healthy control (15 cases) were determined by flowcytometry.Results Th17 cells were significantly higher in the long-term group compared with short-term group and healthy control group(P ＜0.01).Tregs in long-term group compared with short-term group were significantly higher (P ＜ 0.01),but the difference was not statistically significant compared with healthy control group (P ＞ 0.05).NK cells were significantly higher in long-term group compared with short-term group and healthy control group (P ＜ 0.01).MDSC were significantly higher in long-term group compared with short-term group and healthy control group (P ＜0.01).Conclusions Th17,Tregs,NK cells and MDSC were significantly higher in long-term survival of liver recipients,which may be related to immune tolerance.

Objective To investigate the safety and feasibility of a simplified protocol combining with mesenchymal stem cell (MSC) in ABO-incompatible (ABO-I) liver transplant patients. Methods Twelve ABO-I liver transplant patient who received the therapy of a simplified protocol combining with MSC in the Third Affiliated Hospital of Sun Yat-sen University between January 2014 and September 2015 were recruited in this prospective study. Ten cases were male and 2 were female, with a mean age of (39±13) years old. The informed consents of all patients or their families were obtained and the local ethical committee approval was received. A immunologic tolerance induction protocol, plasma exchange + rituximab + intravenous immunoglobin + MSC (simplified protocol combining with MSC and without splenectomy and graft local infusion), was used to prevent the antibody-mediated rejection (AMR) after liver transplantation (LT). The perioperative condition and postoperative outcome of the patients were observed. Results Three death cases were observed after LT including 2 cases died of multiple organ failure and 1 of gastrointestinal hemorrhage. The other cases survived. Two cases developed acute cellular rejection and no AMR case was observed. Biliary complication was observed in 3 cases, hepatic artery stenosis in 1 case and infection in 6 cases. Conclusion The simplified protocol combining with MSC is safe and effective in preventing the AMR after ABO-I LT.

OBJECTIVETo assess the association of vitamin D receptor (VDR) gene Fok I and Bsm I polymorphisms with dyslipidemia in elderly male patients with type 2 diabetes of Han nationality.

METHODSA total of 328 elderly male residents of Han nationality in Beijing, including 237 type 2 diabetic patients and 91 healthy control subjects, were enrolled in this study. The diabetic patients were divided into non-dyslipidemia group (DO group, n=134) and dyslipidemia group (DH group, n=103). All the participants were genotyped for Fok I and Bsm I polymorphisms in VDR gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing technology, and the results were compared with their clinical characteristics.

RESULTSFor Fok I, the frequency of F allele was significantly higher in the diabetic patients than in the control group (Χ(2)=3.873, P=0.049, OR=1.439, 95% CI: 1.001-2.071). In the dominant model, the frequency of FF genotype was significantly higher in the diabetic group (Χ(2)=5.057, P=0.025, OR=1.756, 95% CI: 1.072-2.875) as well as in DH group (Χ(2)=6.168, P=0.013, OR=2.06, 95% CI: 1.161-3.663) than in the control group. There was no significant differences in the genotype frequency or allele distribution in other paired groups (P>0.05). Compared with Ff + ff genotype, FF genotype was associated with a significantly decreased average diastolic blood pressure (P=0.039) but significantly increased postprandial blood glucose (P=0.035), triglycerides (P=0.049) and uric acid (P=0.031). No significant difference was detected in genotype frequency or allele distribution of Bsm I polymorphisms between the groups (P>0.05); serum creatinine levels were significantly higher in bb genotype than in BB + Bb genotype group (P=0.011).

CONCLUSIONVDR gene Fok I polymorphisms may be a risk factor for dyslipidemia in elderly male patients with type 2 diabetes among Chinese Han population, where Bsm I polymorphisms are not associated with diabetic dyslipdiemia.

Aged ; Alleles ; Blood Glucose ; Blood Pressure ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; genetics ; Dyslipidemias ; genetics ; Ethnic Groups ; Genotype ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Receptors, Calcitriol ; genetics ; Risk Factors ; Triglycerides ; blood

Objective To assess the association of vitamin D receptor (VDR) gene Fok I and Bsm I polymorphisms with dyslipidemia in elderly male patients with type 2 diabetes of Han nationality. Methods A total of 328 elderly male residents of Han nationality in Beijing, including 237 type 2 diabetic patients and 91 healthy control subjects, were enrolled in this study. The diabetic patients were divided into non-dyslipidemia group (DO group, n=134) and dyslipidemia group (DH group, n=103). All the participants were genotyped for Fok I and Bsm I polymorphisms in VDR gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing technology, and the results were compared with their clinical characteristics. Results For Fok I, the frequency of F allele was significantly higher in the diabetic patients than in the control group (χ2=3.873, P=0.049, OR=1.439, 95%CI:1.001-2.071). In the dominant model, the frequency of FF genotype was significantly higher in the diabetic group (χ2=5.057, P=0.025, OR=1.756, 95%CI:1.072-2.875) as well as in DH group (χ2=6.168, P=0.013, OR=2.06, 95%CI:1.161-3.663) than in the control group. There was no significant differences in the genotype frequency or allele distribution in other paired groups (P>0.05). Compared with Ff + ff genotype, FF genotype was associated with a significantly decreased average diastolic blood pressure (P=0.039) but significantly increased postprandial blood glucose (P=0.035), triglycerides (P=0.049) and uric acid (P=0.031). No significant difference was detected in genotype frequency or allele distribution of Bsm I polymorphisms between the groups (P>0.05); serum creatinine levels were significantly higher in bb genotype than in BB + Bb genotype group (P=0.011). Conclusion VDR gene Fok I polymorphisms may be a risk factor for dyslipidemia in elderly male patients with type 2 diabetes among Chinese Han population, where Bsm I polymorphisms are not associated with diabetic dyslipdiemia.

Objective To assess the association of vitamin D receptor (VDR) gene Fok I and Bsm I polymorphisms with dyslipidemia in elderly male patients with type 2 diabetes of Han nationality. Methods A total of 328 elderly male residents of Han nationality in Beijing, including 237 type 2 diabetic patients and 91 healthy control subjects, were enrolled in this study. The diabetic patients were divided into non-dyslipidemia group (DO group, n=134) and dyslipidemia group (DH group, n=103). All the participants were genotyped for Fok I and Bsm I polymorphisms in VDR gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing technology, and the results were compared with their clinical characteristics. Results For Fok I, the frequency of F allele was significantly higher in the diabetic patients than in the control group (χ2=3.873, P=0.049, OR=1.439, 95%CI:1.001-2.071). In the dominant model, the frequency of FF genotype was significantly higher in the diabetic group (χ2=5.057, P=0.025, OR=1.756, 95%CI:1.072-2.875) as well as in DH group (χ2=6.168, P=0.013, OR=2.06, 95%CI:1.161-3.663) than in the control group. There was no significant differences in the genotype frequency or allele distribution in other paired groups (P>0.05). Compared with Ff + ff genotype, FF genotype was associated with a significantly decreased average diastolic blood pressure (P=0.039) but significantly increased postprandial blood glucose (P=0.035), triglycerides (P=0.049) and uric acid (P=0.031). No significant difference was detected in genotype frequency or allele distribution of Bsm I polymorphisms between the groups (P>0.05); serum creatinine levels were significantly higher in bb genotype than in BB + Bb genotype group (P=0.011). Conclusion VDR gene Fok I polymorphisms may be a risk factor for dyslipidemia in elderly male patients with type 2 diabetes among Chinese Han population, where Bsm I polymorphisms are not associated with diabetic dyslipdiemia.

Objective To investigate the correlation between serum osteocalcin and type 2 diabetes mellitus (T2DM) in elderly patients.Methods A total of 55 T2DM patients (diabetic group) and 50 non-diabetic subjects (control group) aged ≥60 years were enrolled in this study.The levels of fasting blood glucose (FBG),glycated hemoglobin (HbAlc),insulin resistance index (HOMA IR),osteocalcin (OC),body mass index (BMI) and bone mineral density (BMD) in groups were compared.The correlations between serum osteocalcin and the above indicators were analyzed.Results The levels of OC and BMD were higher in control group than in diabetic group [(11.2±3.2)μg/Lvs.(4.1±3.0)μg/L,(-1.3±0.3) vs.(-2.6±0.5),respectively,both P＜0.05].The levels of FBG,HbAlc,FINS,HOMA-IR were lower in control group than in diabetic group [(4.7±2.0) mmol/L vs.(9.4±2.1) mmol/L],[(4.8±1.5) ％ vs.(7.6±1.6)％,(7.4±3.2) U/L vs.(23.7±3.0) U/L,(1.5±0.7) vs.(9.9±1.2),respectively,all P＜0.05].Serum osteocalcin concentration was negatively correlated with the levels of FBG,HbAlc,FINS and HOMA-IR in elderly patients with T2DM (r=-0.739,-0.713,-0.613,-0.092,all P＜0.01).Conclusions Serum osteocalcin concentration is correlated with the levels of blood sugar and insulin resistance index in elderly patients with T2DM.A further study on the correlation between osteocalcin and T2DM may provide a new target for the treatment of type 2 diabetes mellitus.