BACKGROUND:To explore the methods for secondary limb revascularization after endovascular repair is one of the most urgent research topics.
OBJECTIVE:To investigate the biological mechanism about the transplantation of autologous peripheral blood stem cel s to lead and perfect the effect of ischemic tissue for secondary revascularization.
METHODS:Forty-two patients with critical low limb ischemia were selected and treated with endovascular repair as first revascularization and transplantation of autologous peripheral blood stem cel s as secondary revascularization. The secondary revascularization was carried out at 3 months after the first blood flow remodeling. The patients receiving secondary revascularization were fol owed for 4 years.
RESULTS AND CONCLUSION:After transplantation of autologous peripheral blood stem cel s, the patients complained that the rest pain of the lower leg was relieved obviously and intermittent claudication distance was significantly lengthened. Limb salvage rate was 100%. Skin temperature index was 1.6±0.3, transcutaneous oxygen pressure was (5.00±1.26) kPa, ankle-brachium index was 0.9±0.2, photoplethysmograpy index was 0.8±0.1, saturation of blood oxygen was (79.4±20.4)%, and digital subtraction angiography score was (1.3±0.2)
points. After transplantation, local blood flow indicators of the low limbs were positively correlated to local symptom indicators of the limbs (0.6

Objective: To investigate the pharmacokinetic and pharmacodynamic changes of Yangyin Tongnao Granules (YTG) in cerebral ischemia reperfusion rats after the compatibility of main effective parts (total alkaloids, total flavonoids, total saponins and total phenolic acids). Methods: By using the orthogonal design to research the main effective parts of YTG, nine different dosage ratios combinations were formed, which were used for oral administration in cerebral ischemia reperfusion rats. High performance liquid chromatography-diode array detector (HPLC-DAD) was used to determine the concentration of puerarin, ferulic acid, and ligustrazine in rat plasma at different time points. The non-compartmental model was used to fit the pharmacokinetic parameters by Drug and Statistics (DAS) 3.2.6 software. The total quantum statistical moment analysis method and comprehensive evaluation method were used to evaluate the total pharmacokinetic characteristics. Meanwhile, the content of superoxide dismutase (SOD) and catalase (CAT) were determined by enzyme-linked immunosorbent assay (ELISA) kits. Finally, the PK-PD model and the quantitative equations between drug concentration and efficacy were obtained. Results: The pharmacokinetic characteristics of puerarin, ferulic acid and ligustrazine in cerebral ischemia-reperfusion rats were different. Total statistical moment and comprehensive score study showed that different combinations had different effects on ACUT, mean retention time (MRT), and comprehensive evaluation. The effective parts inhibited the reduction of oxidation indexes such as SOD and CAT. Sigmoid-Emax models were adopted in all PK-PD models, and the fitting results had a good correlation with the measured data. The R values were more than 0.85. Conclusion: Compatibility of YTG activity parts had a certain effect on their pharmacokinetic behaviors and antioxidation in model rats. The total quantum statistical moment analysis and comprehensive evaluation method can be used to study the pharmacokinetics of multi-component traditional Chinese medicine prescriptions. PK-PD model could be used to predict and evaluate the correlation between pharmacokinetics and pharmacodynamics of traditional Chinese medicine prescriptions.

Objective To investigate the efficiency of autologous transplantation of peripheral blood stem cell for treatment of patients with chronic lower limb ischemia. Methods Forty-six patients with chronic lower limb ischemia were treated by autologous peripheral blood stem cell transplantation. Results Fortytwo patients had pain relief on legs, and cold and cool feelings in lower limbs disappeared. Thirty-five patients had pain relief on feet, and 29 of them had disappearance of cold and cool feelings. Due to necrosis in middle and lower part of leg, 4 patients took extremity amputation after 4 weeks of the transplantation. In the 42 patients who kept their legs 3 months after transplantation, the distance of anginacruris extended from (87. 45 ±41.22) m to (348.52 ± 147.24) m, skin temperature increased from(28.52 ±0.51 ) ℃ to(33.56±0.62) ℃ ,and ankle-brachial index (ABI) increased from(0.48 ±0.06)to(0.75 ±0.07). There were statistical differences, and the scores of the distance of anginacruris, skin temperature, and ABI after transplantation were better than before. six months after transplantation autobiography of lower extremity was used, and neonatal lateral vessels of different degrees were found in 37 patients. Conclusion Autologous transplantation of peripheral blood stem cell is a simple, safe, and effective method, especially in treating patients with lower limb ischemia in which no arterial reconstruction is feasible.

Objective To explore the expression of(hyopxia inducible factor-α,HIF-1α)and gax genes on the intimal hyperplasia for venous autografts in rat.Methods Sixteen Wistar rats were randomLy divided into 2 groups,a control group and an experimental group,8 rats in each group.A rat model of venous autografts was established by transplanting the right external jugulars vein into right common carotid artery in 16rats.The transplanted vein in the experimental group was immersed in solution with recombinant adeno-associated virus(rAAV)and HIF-1α by incubation for 45 minutes at 0℃-4℃ before anastomosis and coated using glue gel in which rAAV-gax added just after anastomosis,the control ones were immersed only in the physiological solution in which rAAV solution without HIF-1α gene added,nothing to be coated except for glue gel only.The vein grafts were taken at 14 days after operation,RT-PCR technique,Western blotting and electric microscopy were used to detect the expression of HIF-1α mRNA and protein,gax mRNA and protein,phenotypic switch of VSMC.Results The expression of HIF-1α mRNA,the experimental group(93.1±22.9)bP,the con trol group(42.7±15.9)bP,P<0.01;the exprossion of gax mRNA,the experimental group(106.9±38.7)bP,the contrd group(50.7±25.8)bP,P<0.01;the exprossion of HIF-1α protein,the experimental group(10.86±2.76)kD,the control group(4.52±1.90)kD,P<0.01;the expression of gax protein,the experimental group(13.65±3.35)kD,the control group(5.40±2.26)kD,P<0.01;VSMC proliferation,the experinental group(21.3±5.4)%,the control group(41.2±8.6)%,P<0.05.Conclusion The present study demonstrated that HIF-1α and gax genes transfection could inhibit proliferation of VSMC and their phenotypic conversion from contractile phenotype to synthetic phenotype in venous autografts.

Child ; Humans ; Middle Ear Ventilation ; Otitis Media ; Otitis Media with Effusion ; surgery ; Recurrence

ObjectiveTo observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.ResultsCompared with control group,the expression of procollagen typeⅠ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).ConclusionsThis result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation.

A new and efficient reaction system has been set up, in which the MseⅠ primers were fluorescent labeled for auto-sequencer. PCR reagents and primers and adapters of MseⅠ and EcoRⅠ, which were synthesized, the AFLP protocol has been modified, and reaction and electrophoresis conditions were optimized, the results obtained can be comparable to that of AFLP fluorescent labeled AFLP kits with less cost.

Objective To investigate the effects of chronic fluorosis on the expressions of matrix metalloproteinase-9 (MMP-9) mRNA and protein and the differentiation and maturation process of bone cell in the osteoclast of bone tissue of rats.Methods According to body weight,thirty-six healthy SD rats(body mass 100-120 g) were divided into three groups by random number table,twelve in each group,half male and half female.The rats of control group were given tap water(NaF ＜ 1 mg/L),and rats of low-fluorine and high-fluorine groups were fed with tap water containing 5 and 50 mg/L NaF to establish chronic fluorosis model.Rats were sacrificed after eight months; the contents of urinary fluoride in 24 hours and bone fluoride were analyzed by fluoride selective electrode.Serum content of tartrate resistant acid phosphatase 5b(TRACP5b)was detected by enzyme-linked immunosorbent assay (ELISA).The paraffin section of bone tissue was stained by hematoxylin-eosin (HE) and pathological morphometry was observed under optical microscope.The protein and mRNA levels of MMP-9 in the osteoclast of bones were detected by immunohistochemistry (IHC) and in situ hybridization (ISH),respectively.Results The differences of fluoride contents of urine and bone in rats were statistically significant between groups(F =400.612,48.229,all P ＜ 0.05).Fluoride contents of urine and bone were increased in lowfluorine and high-fluorine groups[(6.09 + 0.56),(7.69 + 0.64)mg/L,(12.65 ± 3.07),(26.53 + 5.88)mg/kg] compared to the control groups[(1.36 ± 0.51)mg/L,(0.67 ± 0.16)mg/kg,all P ＜ 0.05],and the fluoride contents of urine and bone were gradually increased with increasing fluoride doses(all P ＜ 0.05).The difference of TRACP5b content in serum was statistically significant between groups (F =9.607,P ＜ 0.05),in low-fluorine and high-fluorine groups,the TRACP5b contents[(1.86 ± 0.13),(1.92 ± 0.22)U/L] were higher than that of control group [(1.57 + 0.20)U/L,all P ＜ 0.05].The pathological examination showed osteosclerosis in fluoride exposed groups.The differences of MMP-9 mRNA and protein expressions were statistically significant between groups (F =365.727,331.382,all P ＜ 0.05).Compared to the control groups(97.22 ± 2.24,78.51 ± 1.16),the expressions of MMP-9 protein(108.18 ± 1.97,119.28 ± 1.76) and mRNA(89.44 ± 2.86,102.14 ± 2.39) were increased(all P ＜ 0.05),and the expressions of MMP-9 mRNA and protein were gradually increased with increasing fluoride doses (all P ＜ 0.05).Conclusions Chronic fluorosis might influence osteoclast differentiation and maturation process through regulating the expression levels of MMP-9 protein and mRNA.

Human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) might be developed as a novel anti-tumor drug due to its selective cytotoxicity in tumor cells. The predicted Macaca mulatta TRAIL (mmTRAIL) is highly homologous to hTRAIL in nucleotide acid as well as amino acid sequence, suggesting that mmTRAIL might induce apoptosis of human cancer cells. However, the cytotoxicity of mmTRAIL in human cancer cells has not been investigated. In this paper, it is reported that the gene encoding mmTRAIL has been cloned by using reverse-transcriptase polymerase chain reaction (RT-PCR) from monkey peripheral blood mononuclear cells (PBMCs) in our laboratory. Subsequently, an expression plasmid was constructed by inserting mmTRAIL gene into pQE30 plasmid. After induction by addition of Isopropyl β-D-1-Thiogalactopyranoside (IPTG), mmTRAIL was expressed. MmTRAIL was recovered from supernatant of sonicated bacteria by Ni-NTA agarose affinity chromatography. SDS-PAGE and gel filtration chromatography demonstrated that mmTRAIL forms trimer in solution. In vitro assays indicated that mmTRAIL was cytotoxic to human COLO205 tumor cells but not to normal cells at low concentration of nanomole. In addition, antitumor effect of mmTRAIL was evaluated in mice bearing human COLO205 tumor xenografts. Intratumorally injected mmTRAIL significantly inhibited growth of tumor grafts. These results suggested that mmTRAIL was valuable as candidate drug for cancer-targeted therapy.
Animals ; Antineoplastic Agents ; Apoptosis ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Leukocytes, Mononuclear ; Macaca mulatta ; Mice ; Plasmids ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Xenograft Model Antitumor Assays

Background Congenital aniridia is a rare bilateral hereditary ophthalmopathy which impact panocular.Researches showed that congenital aniridia can be caused by different mutation locus of PAX6 genes,and the mutations are multifarious.Objective This study was to detect and anaiyze the mutations of a Chinese family with congenital aniridia by using targeted sequence capture sequencing and direct Sanger sequencing.Methods This study was approved by Ethic Committee of the First Affiliated Hospital of Zhengzhou University and followed Declaration of Helsinki.Written informed consent was obtained from subjects or their custodians before any related medical examination.A cross-sectional study was performed.A Chinese congenital aniridia family was included at the First Affiliated Hospital of Zhengzhou University in March,2016.All the family members received systemic medical examinations including nervous system and oral glucose tolerance test and then the ocular examinations were carried out.The periphery blood of 10 ml was collected from the members for genomic DNA extraction.Targeted sequence capture sequencing was performed on the DNA of proband to screen out the suspicious mutant locus.The mutation was verified by comparing the Sanger direct sequencing results from all family members.Results A total of 3 generations of 9 members were included in this congenital aniridia pedigree,and the Ⅰ 1 was dead without eye abnormality.Three patients (Ⅱ2 and her children Ⅲ1,Ⅲ2) and 5 normal family members were determined,showing an autosomal dominant inheritance pattern.No abnormal signs were found in nervous system and oral glucose tolerance test in the families.The reduce of visual acuity,ocular hypertension (21 mmHg),absence of biocular iris,opacification of corneal stroma,horizontal nystagmus,hapoplasia of fovea were found in all the sufferers.In addition,the ptosis of the left eye,congenital cataract of the right eye in Ⅱ 2 patient as well as biocular cataract and subluxation of lenses also were exhibited.The c.183C＞A mutation of the PAX6 gene was screened out to be a possible pathogenic mutation.The result of Sanger direct sequencing in the families verified a co-segregation of this mutation with mutant phenotypes.Conclusions PAX6 gene c.183C ＞A,a rare mutation in Chinese population,is a virulence mutation site in this aniridia family.