Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective:To study the effect of Xuebijing on inflammatory response and prognosis in patients with septic shock.Methods:A prospective randomized controlled study was conducted. Eighty septic shock patients admitted to department of critical care medicine of the First Affiliated Hospital of Zhengzhou University from January to December in 2019 were enrolled. The enrolled patients were divided into Xuebijing group and control group by randomized number table method, with 40 cases in each group. Both groups were strictly followed the guidelines for the diagnosis and treatment of septic shock to take comprehensive treatment measures against sepsis. On this basis, Xuebijing group received intravenous 100 mL Xuebijing injection twice a day for 7 days. Baseline data of enrolled patients were recorded. The levels of interleukin-6 (IL-6), procalcitonin (PCT), C-reactive protein (CRP) and heparin binding protein (HBP) were measured before treatment and 3, 7 and 10 days after treatment. Mechanical ventilation time, the length of intensive care unit (ICU) stay, total hospitalization time and 28-day mortality were recorded. The differences of every indicator between the two groups were compared. Independent risk factors affecting patient prognosis were analyzed by binary Logistic regression.Results:① There was no significant difference in baseline data such as gender, age, infection site, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) and sequential organ failure score (SOFA) between the two groups. ② The levels of serum inflammatory factors in both groups showed a decreasing trend after treatment. Compared with the control group, IL-6 and HBP in the Xuebijing group significantly decreased on day 7 [IL-6 (ng/L): 66.20 (16.34, 163.71) vs. 79.81 (23.95, 178.64), HBP (ng/L): 95.59 (45.23, 157.37) vs. 132.98 (73.90, 162.05), both P < 0.05]; on day 10, PCT, CRP, IL-6 and HBP significantly decreased [PCT (μg/L): 1.14 (0.20, 3.39) vs. 1.31 (0.68, 4.21), CRP (mg/L): 66.32 (19.46, 115.81) vs. 89.16 (20.52, 143.76), IL-6 (ng/L): 31.90 (13.23, 138.74) vs. 166.30 (42.75, 288.10), HBP (ng/L): 62.45 (29.17, 96.51) vs. 112.33 (58.70, 143.96), all P < 0.05]. ③ Compared with the control group, mechanical ventilation time and the length of ICU stay were significantly shortened and the total hospitalization expenses were significantly reduced in Xuebijing group [mechanical ventilation time (hours): 57.0 (0, 163.5) vs. 168.0 (24.0, 282.0), the length of ICU stay (days): 8.80±4.15 vs. 17.13±7.05, the total hospitalization expenses (ten thousand yuan): 14.55±7.31 vs. 20.01±9.86, all P < 0.05]. There was no significant difference in 28-day mortality and the total hospitalization time [28-day mortality: 37.5% vs. 35.0%, the total hospitalization time (days): 13.05±8.44 vs. 18.30±9.59, both P > 0.05]. ④ Patients were divided into death and survival groups according to the prognosis, and univariate analysis showed that white blood cell (WBC), neutrophil percentage (NEU%), CRP, lactic acid (Lac), APACHEⅡ score, IL-6, HBP were the factors influencing the prognosis of patients. The above indicators were further analyzed by Logistic regression, which showed that CRP, IL-6, and APACHE Ⅱ score were independent risk factors for prognosis [odds ratio ( OR) was 1.007, 1.828, 1.229, all P < 0.05]. Conclusions:Combined with Xuebijing to treat septic shock can reduce the body's inflammatory response to a certain extent, thereby reducing the time of mechanical ventilation, shortening the stay of ICU and reducing the total cost of hospitalization. But it cannot reduce the 28-day mortality of patients with septic shock.

Objective The risk of current pancreaticojejunostomy is carefully considered from the perspective of the morphology of remnant pancreas,and we aimed to discuss the clinical outcomes of selecting different pancreaticojejunostomy techniques based on pancreatic morphology.Methods This was a prospective cohort study.The histopathology of remnant pancreatic tissues was categorized into four types based on preoperative radiological images and intraoperative palpation:Type Ⅰ:pancreas with hard texture in palpation,pancreatic atrophy,dilated pancreatic duct larger than 5 mm and remnant pancreatic surface ＜3 cm;Type Ⅱ:pancreas with hard texture in palpation,pancreatic atrophy and mild dilatation of pancreatic duct with the diameter of 3-5 mm and remnant pancreatic surface ＜3 cm;Type Ⅲ:pancreas with slightly hard texture,no atrophy,and normal or slightly dilated pancreatic duct with the diameter of 3-5 mm and remnant pancreatic surface ≥3 cm;Type Ⅳ:pancreas with soft texture,normal morphology and pancreatic duct.Results From January 2008 to August 2017,116 consecutive patients underwent pancreaticoduodenectomy in our center.Among them,10 patients with type Ⅰ underwent classic pancreatic ductal mucosa to mucosa anastomosis.19 patients with type Ⅱ underwent classic end to end invaginated pancreaticojejunostomy.45 patients with type Ⅲ underwent classic end to end invaginated pancreaticojejunostomy with overlapping U sutures;42 patients with type Ⅵ underwent total invaginated pancreaticojejunostomy.The post-operative pancreatic fistula occurred in 6 patients (5.2％) with one patient died.Postoperative bleeding occurred in 10 patients (8.6％),and gastroparesis occurred in 22 patients (19.0％).Overall complication rate was 33.6％.Conclusions Classification of pancreatic morphology based on preoperative radiological images and intraoperative palpation and the selection of corresponding pancreaticojejunostomy technique is theoretically rational and has the advantage of potentially reducing the risk of remnant pancreatic tissue.

OBJECTIVEThe aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells (MSCs) with tumor cells can promote tumor angiogensis.

METHODSHuman glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene. Bone marrow mesenchymal stem cells (MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression. Then the two kinds of cells were co-cultured in vitro. At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors. The co-cultured cells, GFP/RFP double positive (yellow) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.

RESULTSAfter five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105. CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas. When MSCs were co-cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7% of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.

CONCLUSIONCell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.

Animals ; Bone Marrow Cells ; physiology ; Cell Communication ; Cell Fusion ; Cells, Cultured ; Glioma ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; Mesenchymal Stromal Cells ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; Neoplasms ; Neovascularization, Pathologic ; Stem Cells ; Transfection ; Transplantation, Heterologous

Objective To establish a new congenic inbred mouse strain carrying and expressing EGFP and Foxn1nugene for cancer research including human glioma as well .Methods According to criterion of GB14923-2010, the male Foxn1nu nude mice backcross the female C57BL/6-Tg (CAG-EGFP) transgenic mice for 10 times, Then identify the phenotype using the methods and equipment as below: fluorescent flashlight and matching glasses; multifunction vivo imager; fluorescence microscopy.Results The congenic inbred mouse strain named Foxn1nu.B6-CAG-EGFP/SU ( Soochow University ) .All the 14 biochemical loci are homozygous and same with Balb/c mouse in addition to the Pep3 loci (“b” type instead of “a” type).Peripheral blood lymphocyte count shows the lymphocytes occupy 15%of nucleated cells;T lymphocytes occupy 0.3%, meet the requirement of inbred strain of EGFP nude mice .Conclusions Established a new congenic inbred strain -Foxn1nu.B6-CAG-EGFP/SU which both express EGFP stably, and own immunodeficiency with lack of T lymphocytes .The phenotype “b” of biochemical loci “Pep3” is the unique characteristic that distinguish SU to Foxn1nu.

Objective The aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells ( MSCs) with tumor cells can promote tumor angiogensis.Methods Human glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene.Bone marrow mesenchymal stem cells ( MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression.Then the two kinds of cells were co-cultured in vitro.At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors.The co-cultured cells, GFP/RFP double positive ( yellow ) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.Results After five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105.CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas.When MSCs were co -cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7%of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.Conclu sion Cell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.

Objective The aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells ( MSCs) with tumor cells can promote tumor angiogensis.Methods Human glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene.Bone marrow mesenchymal stem cells ( MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression.Then the two kinds of cells were co-cultured in vitro.At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors.The co-cultured cells, GFP/RFP double positive ( yellow ) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.Results After five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105.CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas.When MSCs were co -cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7%of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.Conclu sion Cell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.

Objective To investigate the correlation of single nucleotide polymorphism of rs3731055 and rs2607775 of xeroderma pigmentosum group C (XPC) and smoking with genetic susceptibility to pancreatic cancer.Methods The clinical data of 214 patients with pancreatic cancer who were admitted to the First and Third Affiliated Hospitals of Xinjiang Medical University from January 2009 to June 2011 and 214 healthly individuals were retrospectively analyzed.The samples of venous blood of 214 patients with pancreatic cancer (case group) and 214 healthy individuals (control group) were analyzed by the Multiplex SNaPshot method.The count data were analyzed using the chi-square test.The association between the single nucleotide polymorphism of rs3731055 and rs2607775 with genetic susceptibility to pancreatic cancer was analyzed using the Logistic regression method.Results Four hundred and twenty-three samples of gene were successfully typed,including 210 in the case group and 213 in the control group.The frequency of G allele of XPC rs3731055 was 75.95％ (319/420) in the case group and 77.00％ (328/426) in the control group,with no significant difference between the 2 groups (x2 =0.12,P ＞ 0.05).The frequencies of genotypes GG,GA and AA were 58.57％ (123/210),34.76％ (73/210) and 6.67％ (14/210) in the case group,and 60.09％ (128/213),33.80％ (72/213) and 6.10％ (13/213) in the control group,with no significant difference between the 2 groups (x2=0.12,P ＞ 0.05).The frequency of C allele of XPC rs2607775 was 87.86％ (369/420) in the case group and 93.43％ (398/426) in the control group,with significant difference between the 2 groups (x2=7.75,P ＜ 0.05).The frequencies of genotypes CC,CG and GG were 77.62％ (163/210),20.48％ (43/210) and 1.90％ (4/210) in the case group,and 86.85％ (185/213),13.15％ (28/213) and 0(0/213) in the control group,with significant difference between the 2 groups (x2=8.54,P ＜ 0.05).Patients with rs2607775 GC genotype were associated with a significantly increased risk of pancreatic cancer compared with patients with rs2607775 CC genotype (adjusted OR =1.81,95％ CI:1.06-3.10,P ＜ 0.05).Patients with rs2607775 GC + GG genotype were associated with a significantly increased risk of pancreatic cancer compared with patients with rs2607775 CC (adjusted OR =1.98,95％ CI:1.16-3.36,P ＜ 0.05).The ratio of patients in the case group who smoked cigarettes ≥ 17 pack years was 25.24％ (53/210),which was significantly higher than 13.15 ％ (28/213) of the control group (x2 =11.37,P ＜ 0.05).The results of univariate analysis showed that patients who smoked cigarettes ≥ 17 pack years had higher risk of getting pancreatic cancer (adjusted OR =2.82,95％ CI:1.27-6.29,P ＜ 0.05).Patients who smoked cigarettes ≥ 17 pack years and with rs2607775 CC also had higher risk of getting pancreatic cancer (adjusted OR =2.87,95％ CI:1.18-6.99,P ＜0.05).No significant gene-environment interaction was observed between rs2607775 GC + GG and smoking ≥ 17 pack years (adjusted OR =3.65,95％ CI:0.67-20.03,P ＞ 0.05).Conclusions The polymorphisms of XPC rs2607775 may play a role in the onset of pancreatic cancer.Patients who smoke cigarettes ≥ 17 pack years are more easily to have pancreatic cancer.There is no interaction between smoking and XPC rs2607775 in influencing the progression of pancreatic cancer.

Objective To systematically review the clinical efficacy of transanastomotic pancreatic ductal stents placement after pancreaticoduodenectomy.Methods According to the Cochrane reviewers handbook (version 5.0 ),literatures were retrived from PubMed,Embase,Cochrane,VIP database,China Biology Medicine disc and CNKI database,and then the quality of the literatures was analyzed.Meta analysis was carried out using the RevMan software ( version 5.0.18 ).A random effects model was adopted,and the results of the meta analysis were presented with odds ratio (OR) and 95％ confidence interval (95％ CI).Results Four randomized controlled trials including 557 patients were retrieved.External stents were used in 160 patients and internal stents in 115 patients.The results of meta analysis showed no significant difference in the rate of fistula,overall postoperative morbidity and mortality between patients who did or did not receive pancreatic stents placement (OR =0.66,0.70,0.63,P ＞ 0.05 ).There were significant differences in the rate of pancreatic fistula and overall postoperative morbidity between patients who received external pancreatic stents placement and those did not receive pancreatic stents placement ( OR =0.48,0.55,P ＜ 0.05 ).There was no significant difference in the mortality rate between patients who received external pancreatic stents placement and those did not receive pancreatic stents placement (OR =0.71,P ＞ 0.05 ).There were no significant difference in the incidence of pancreatic fistula,overall postoperative morbility and mortality between patients who received internal pancreatic stents placement and those did not receive pancreatic stents placement ( x2 =0,0.75,2.11,P ＞ 0.05 ).Conclusions External pancreatic stents placement after pancreaticoduodenectomy can reduce the incidence of postoperative complications.The effects of internal pancreatic stents placement need to be proved by further highquality prospective randomized trials.