Objective: To figure out the clinical application value of indocyanine green (ICG) lymphangiography in supermicrosurgical lymphaticovenular anastomosis. Methods: A total of 6 supermicrosurgical lymphaticovenular anastomosis with intraoperative ICG lymphangiography were performed during April 2015 to May 2017 and were analyzed retrospectively. All the cases are female (range from 30 to 54 years old, median of 46.5 years old), including 3 cases for prevention and 3 cases for treatment of lymphedema. Results: A total 6 supermicrosurgical lymphaticovenular anastomosis were performed with intraoperative ICG lymphangiography to make sure the influx of lymph fluid to the vein. During the median of 23 months follow-up (range from 7 to 32 months), the 3 preventive cases did not show upper limb lymphedema and the 3 theraputic cases were relieved at different levels. Conclusions Intraoperative ICG lymphangiography can provide real-time information to locate suitable lymph vessels and ascertain the anastomotic patency in supermicrosurgical lymphaticovenular anastomosis, thus improve the operation effectiveness.

Objective: This article provides an overview of our experience using indocyanine green angiography (ICGA) in breast reconstruction with abdominal flap to ascertain the application value of ICGA and its usage in decreasing postoperative complications. Methods: A total of 21 breast reconstructions with intraoperative ICGA were analyzed retrospectively, including 7 bilateral deep inferior epigastric perforator (DIEP) flaps, 5 pedicled transverse rectus abdominis myocutaneous (TRAM) flaps with contralateral free TRAM flaps, 4 pedicled TRAM flaps with contralateral DIEP flaps, 3 unilateral DIEP flaps and 2 unilateral pedicled TRAM flaps. According to different breast reconstruction methods, ICGA were applied respectively after flap harvesting and vessel anastomosis, in order to evaluate the blood supply of flaps and vessel perfusion. Results: A total of 52 ICGA were performed and recorded intraoperatively without any indocyanine green-associated complications. The operation methods were modified according to ICGA findings in 6 of 21 cases. The distal part of flaps were discarded due to poor perfusion in 2 cases (1 DIEP flap and 1 TRAM flap), additional free vessel anastomosis were needed in 2 cases to ensure sufficient blood supply, 2 vascular complication including 1 vascular occlusion and 1 vascular thrombosis were found and managed in time. During the follow-up (range from 3 to 30 months, median of 16 months), no vascular crisis was reported. All flaps survived satisfactorily without partial or whole flap necrosis or wound infection. Conclusions Intraoperative ICGA can provide real-time information of flap′s blood supply and vessel perfusion to evaluate the conditions of flaps and vascular anastomosis, which can help surgeons take actions accordingly to increase the successful rate of breast reconstruction.

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L β-glycerophosphate and alkalized by 7.4% NaHCO₃ to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β₃ subunit(LTCC β₃) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC β₃ were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC β₃ were higher in high phosphorus+ pH7.7 group (all P<0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P<0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P<0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P<0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P<0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P<0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P<0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P<0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P<0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P<0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P<0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P<0.05). Conclusion Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β₃ subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

Objective To explore the effect of different pH values on calcification of rat vascular smooth muscle cells (VSMCs) through bone morphogenetic protein (BMP)-2 signaling pathway. Methods Healthy male SD rats aged 5-8 weeks were selected in the study. VSMCs from rat thoracic aorta were cultured in vitro, and then identified by immunocytochemistry. The VSMCs were randomly divided into 4 groups by random sampling method:normal group (pH 7.4), pH7.4+high phosphorus group, pH 7.1+high phosphorus group, and pH 7.7+high phosphorus group. Calcium deposition and alkaline phosphatase (AKP) activity were measured by alizarin red staining and enzyme linked immunosorbent assay. The expressions of BMP-2, Smad1 and Runx2 mRNA were detected by RT-PCR. Results Compared with the control group, the calcification staining was increased in pH 7.4+high phosphorus group, calcium content was increased and expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were also increased (P<0.05). While compared with the pH 7.4+high phosphorus group, calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were decreased in pH 7.1+high phosphorus group (P<0.05). The calcification staining, calcium content, expressions of BMP-2, Smad1, Runx2 mRNA and AKP activity were increased in pH 7.7+high phosphorus group (P<0.05). Conclusion The extracellular acidic environment (pH 7.1) can inhibit high-phosphotus-induced VSMCs calcification, whereas extracellular alkaline environment (pH 7.7) induce high-phosphotus-induced VSMCs calcification. The mechanism is presumably that VSMCs calcification is induced by influencing BMP-2 pathway, which may be mediated by VSMCs phenotype transdifferentiation of BMP-2 signaling pathway.

Objective To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). Methods We selected 140 CCRCC patients and 130 healthy controls in this case?control study. Genotype of single nucleotide polymorphism ( rs16917496) at the miR?502 binding site in the 3′UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. Results In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively. The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group ( P<0.05) . Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC ( P<0. 05 ) . CT and TT genotypes of rs16917496 at the miR?502 binding site of the SET8 gene were associated with expression of SET8. Conclusions Genotype of the SNP rs16917496 at the miR?502 binding site in the 3′ untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.

Objective To investigate the relationship between single nucleotide polymorphism of SET8 gene and the risk of clear cell renal cell carcinoma (CCRCC). Methods We selected 140 CCRCC patients and 130 healthy controls in this case?control study. Genotype of single nucleotide polymorphism ( rs16917496) at the miR?502 binding site in the 3′UTR of SET8 mRNA in the CCRCC patients and healthy controls was tested and the association between genotype and risk of cancer was assessed. The expression of SET8 was determined by immunohistochemistry and the relationship between expression of SET8 and genotype of rs16917496 was analyzed. Results In the control group, CC, CT and TT genotypes were found in 30, 32 and 68 persons, respectively, while in the CCRCC patients, CC, CT and TT genotypes were found in 14 , 47 and 79 cases, respectively. The frequencies of rs16917496 CT and TT genotypes in the CCRCC group were significantly higher than those in the control group ( P<0.05) . Compared with the CC genotype, patients with CT and TT genotypes were more susceptible to develop CCRCC ( P<0. 05 ) . CT and TT genotypes of rs16917496 at the miR?502 binding site of the SET8 gene were associated with expression of SET8. Conclusions Genotype of the SNP rs16917496 at the miR?502 binding site in the 3′ untranslated region of the SET8 gene is associated with the expression of SET8 protein. Analysis of genetic polymorphisms in miRNA binding sites may help to identify the subgroups of population susceptible to CCRCC.

Objective To explore the effect and mechanism of magnesium on calcification induced by hyperphosphate.Methods Vascular smooth muscle cells (VSMCs) were primarily cultured in vitro and induced calcification by β-glycerophosphate (β-GP).VSMCs were randomly divided into control group,high phosphorus group (10 mmol/L β-GP),magnesium intervèntion group(10 mmol/L β-GP + 3 mmol/L MgSO4) and 2-aminoethoxy-diphenylborate (2-APB,an inhibitor of magnesium transporter) intervention group(10 mmol/L β-GP+3 mmol/L MgSO4+ 10-4 mol/L 2-APB).Calcium deposition and alkaline phosphatase (ALP) activity were measured by alizarin red staining,quantification of calcium and euzyme linked immunosorbent assay.RT-PCR and Western blotting were used to observe the expression of core binding factor α-1 (Cbfα-1) mRNA and protein,respectively.In vivo,male Sprague-Dawley rats (n=24) were randomly divided into control group (methylcellulose+high phosphorous diet),vascular calcification group (adenine suspension + high phosphorous diet),high magnesium intervention group(adenine suspension+high phosphorous and magnesium diet).The aortic pulse wave velocity (PWV) was measured,and vascular calcification was determined by von Kossa stain and quantification of calcium.Cbfα-1 in aortic was measured by immunohistochemistry.Results In vitro,compared with high phosphorus group,calcification,ALP activity (P ＜ 0.05) and Cbfα-1expression in VSMCs were significantly decreased in magnesium intervention group after incubation for 14 days,but the addition of 2-APB might inhibit the protective effect of magnesium on VSMCs.Dynamic observation of Cbfα-1 showed that magnesium significantly inhibited the expression of Cbfα-1 (P ＜ 0.05) on the third day and the inhibitory role was obviously increased in a time-dependent manner.Consistent with the findings in vitro,the aortic PWV,calcification were all significantly reduced (P ＜ 0.05) in high magnesium intervention group with high serum magnesium level,when compared with vascular calcification group.Immunohistochemistry showed that hypermagnesemia downregulated obviously the expression of Cbfα-1 induced by hyperphosphatemia(P ＜ 0.05).Conclusion Magnesium protects against vascular calcification by inhibiting osteogenic differentiation of VSMCs.

Objective To explore the effect of vitamin K2 on β-glycerophosphate(β-GP)-induced rat vascular smooth muscle cells (VSMCs) calcification and and the mechanism.Methods VSMCs were obtained from rat aortic,and identified by immunocytochemistry,then randomly divided into control group,high phosphorus group,vitamin K2 group (the group was settled three subgroups according to the concentration of vitamin K2 based on the high phosphorus medium,namely 10 μmol/L,25 μmol/L,50 μmol/L) and noggin (bone morphogenetic protein pathway inhibitor) group.Calcification was visualized by Alizarin red staining,calcium load in cells was quantified by o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured after stimulating 14 days,gene expressions of bone morphogenetic protein-2 (BMP-2),SMAD1,SMAD7 and Runx2 mRNA were detected by RT-PCR,Runx2 protein levels was detected by Western blotting after stimulating 3 days.Results Compared with the cells in control group,high phosphorus induced cell calcification,increased ALP activity,up-regulated the expression of BMP-2,SMAD1,Runx2 mRNA (P ＜ 0.05) and down-regulated the expression of SMAD7 (P ＜ 0.01),while compared with high phosphorus group,the calcium deposition,ALP activity and the expression of BMP-2,SMAD1,Runx2 mRNA were remarkably reduced in a dose-dependent manner by treatment with vitamin K2 (P ＜ 0.05) and the expression of SMAD7 was increased (P ＜ 0.01).Compared with high phosphorus group,SMAD1 and Runx2 expression in noggin group were remarkably reduced(P ＜ 0.01).Conclusion Vitamin K2 inhibits β-glycerophosphate-induced VSMCs calcification which correlates with the suppression of the expression of osteoblast markers through the down-regulation of bone morphogenetic protein pathway.

BACKGROUNDThe mitochondrial displacement loop (D-loop) accumulates mutations and single nucleotide polymorphisms (SNPs) at a higher frequency than other regions of mitochondrial DNA (mtDNA). We previously identified disease risk-associated SNPs in the D-loop of chronic kidney disease (CKD) patients; in this study, we investigated the association of age-at-onset and D-loop SNPs in CKD patients.

METHODSThe D-loop region of mtDNA was sequenced in 119 CKD patients attending the Fourth Hospital of Hebei Medical University between 2002 and 2008. The age-at-onset curve of the CKD patients was calculated using the Kaplan-Meier method at each SNP site, and compared using the log-rank test.

RESULTSThe mean age of 119 CKD patients was (55.6±14.2) years, and 56.3% were males. The mean estimated glomerular filtration rate (eGFR) was (81.2±12.4) ml×min(-1)×1.73 m(-2), with 79.8% (n = 95) of patients having an eGFR <60 ml×min(-1)×1.73 m(-2). All participants had an eGFR >30 ml×min(-1)×1.73 m(-2). The age-at-onset for CKD patients who smoked was significantly lower than that of non-smoking CKD patients. The SNP sites of nucleotides 150C/T were identified for their association with age-at-onset using the log-rank test. The age-at-onset of patients with the minor allele T genotype was significantly lower than that of patients with the C genotype at the 150 SNP site (P = 0.010).

CONCLUSIONSGenetic polymorphisms in the D-loop appear to be predictive markers for age-at-onset in CKD patients. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop may help identify CKD patient subgroups at high risk of early onset disease.

Adult ; Aged ; DNA, Mitochondrial ; genetics ; Female ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Renal Insufficiency, Chronic ; genetics

Recent studies suggest that glial cells play an impor-tant role in nervous system. Like astrocytes in the central nervous system,satellite glial cells( SGCs) also participate in the physio-logical and pathological processes of the peripheral nervous sys-tem. SGCs affect neuronal functions through neuro-glial interac-tions. In this review,we summarize the current understanding of how SGCs affect the function of neurons.