OBJECTIVETo observe the effect of sensory neuropeptide substance P combined with epidermal stem cells (ESC) on wound healing and nerve regeneration in diabetic rats.

METHODSESC that had been isolated from SD rats were identified and cultured in vitro, and they were inoculated onto nourishing layer of amniotic membrane to construct amniotic membrane-ESC. Four full-thickness skin wounds were produced on the back of each of 48 diabetic rats. The resulted 192 wounds were randomly divided into ESC + substance P group, ESC group, substance P group, and control group according to the lottery method, with 48 wounds in each group. Wounds in ESC + substance P group and ESC group were transplanted with amniotic membrane-ESC, and those in substance P group and control group were transplanted with amniotic membrane. After transplantation, 250 µL substance P in the concentration of 1 × 10(-7) mol/L was injected around and into the middle of the wounds in ESC + substance P group and substance P group, 2 times a day, and continued for 4 days, while 250 µL PBS solution was injected in the above-mentioned position in ESC group and control group as control, 2 times a day, and continued for 4 days. On post injury day (PID) 4, 7, 10, 14, 17, and 23, the wound healing rate (with 8 wounds at each time point) was observed and determined, and changes in wound tissue structure were observed with HE staining. On PID 4, 7, and 10, collagen distribution in wound tissue was observed with Masson staining, and type I and type III collagen deposition in wound tissue was respectively observed after immunohistochemical staining. The distribution of protein gene product 9.5 (PGP 9.5) and regeneration of substance P positive nerve fibers in wound tissue were observed with immunohistochemical staining on PID 14 and 23. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The wound healing rate in ESC + substance P group reached 100.0% on PID 14, which was obviously earlier than that in ESC group, substance P group, and control group, healing was respectively observed on PID 17, 17, and 23. The wound healing quality in ESC + substance P group was better than that in the other three groups as shown by HE staining. (2) On PID 10, collagen that was darkly stained and widely distributed was observed in wound tissue of ESC + substance P group and substance P group, while collagen in the other two groups was lightly stained and narrowly distributed. Deposition quantity of type I collagen gradually increased, and that of type III collagen gradually decreased in the wounds of each group over time. On PID 4, 7, and 10, distribution amount of type I collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.72, 118.21, 26.71, P values all below 0.01) and control group (with t value respectively 44.37, 22.76, 30.32, P values all below 0.01), while there was no significance between ESC + substance P group and substance P group. On PID 4, 7, and 10, distribution amount of type III collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.27, 28.68, 14.51, P values all below 0.01) and control group (with t value respectively 35.68, 22.52, 22.24, P values all below 0.01). (3) A large amount of PGP 9.5 and regeneration of substance P positive nerve fibers, and some peripheral nerve fibers in deep wound extending to epidermis were observed in wound tissue of ESC + substance P group and substance P group. A small amount of PGP 9.5 and regeneration of substance P positive nerve fibers without peripheral nerve fibers extending to epidermis were observed in deep wound tissue of ESC group and control group. On PID 14, 23, ratios of area of PGP 9.5 positive nerve fiber in the wounds of ESC + substance P group were (3.86 ± 0.25)% and (7.03 ± 0.28)%, and they were significantly higher than those of ESC group [(1.48 ± 0.30)%, (3.01 ± 0.43)%, with t value respectively 23.95, 30.27, P values all below 0.01] and control group [(1.46 ± 0.23)%, (2.84 ± 0.29)%, with t value respectively 27.35, 40.32, P values all below 0.01]. On PID 14, 23, ratios of substance P positive nerve fiber area in the wounds of ESC + substance P group were (2.01 ± 0.14)% and (1.19 ± 0.11)%, which were obviously higher than those of ESC group [(0.85 ± 0.17)%, (1.34 ± 0.21)%, with t value respectively 20.50, 2.60, P < 0.05 or P < 0.01] and control group [(0.74 ± 0.15)%, (1.30 ± 0.17)%, with t value respectively 23.98, 2.41, P < 0.05 or P < 0.01].

CONCLUSIONSJoint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.

Animals ; Diabetes Mellitus, Experimental ; pathology ; Epithelial Cells ; cytology ; Nerve Regeneration ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; Substance P ; pharmacology ; therapeutic use ; Wound Healing

OBJECTIVETo investigate the expressions of vasohibin-1 and MACC1 in lung squamous cell carcinoma (LSCC) and their associations with the clinicopathological characteristics of the patients.

METHODSThe expressions of vasohibin-1 and MACC1 proteins were examined with immunohistochemistry in 160 LSCC tissues and 80 normal lung tissues.

RESULTSThe positivity rates of vasohibin-1 and MACC1 proteins were 59.4% and 11.3% in LSCC tissues, respectively, which were significantly higher than the rates in normal lung tissues (57.5% and 8.8%, respectively; P<0.05). The expressions of vasohibin-1 and MACC1 proteins were significantly correlated with the tumor grades, lymph node metastasis, and TNM stages (all P<0.05). Spearman correlation analysis indicated a positive correlation between vasohibin-1 expression and MACC1 expressions (P<0.001). Kaplan-Meier survival analysis showed that LSCC patients with a positive expression of vasohibin-1 had significantly shorter overall survival time than those negative for vasohibin-1; the overall survival time was also significantly shorter in patients positive for MACC1 than in those negative for MACC1 (both P<0.05). Multivariate COX regression analysis indicated that positive expressions of vasohibin-1 and MACC1 protein and TNM stage were independent prognostic factors of LSCC.

CONCLUSIONAberrant expressions of vasohibin-1 and MACC1 may participate in the development and promote invasion and metastasis of LSCC. The combined detection of vasohibin-1 and MACC1 expression may provide important evidence for predicting the progression and prognosis of LSCC.

OBJECTIVETo prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes.

METHODSShort hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR.

RESULTSThe IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01).

CONCLUSIONCYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.

Apoptosis ; drug effects ; genetics ; Cell Line ; Cell Survival ; drug effects ; genetics ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Genetic Vectors ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lentivirus ; genetics ; RNA Interference ; Trichloroethylene ; toxicity

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.
Antigens, CD34 ; blood ; Blotting, Western ; Cell Differentiation ; DNA-Binding Proteins ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; enzymology ; Humans ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

To evaluated the feasibility of preventing infection after high dose chemotherapy and radiotherapy using the granulocytes derived from differentiated from hematopoietic stem/progenitor cells ex vivo, human CD34-positive cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS), and the cells committedly differentiated with hematopoietic cytokines (SCF + IL-3 + IL-6 + G-CSF) in a liquid culture system. The expanded cell number, ratio of the viable cells, chromosome and phenotype of the differentiated cells and safety analysis of expanded cells were detected by using cell count, trypan blue exclusion test, karyotype analysis, flow cytometry and tumorigenic model of nude mice, respectively. The results showed that the combination of cytokines increased cell number by (1006.4 +/- 103.2) folds and flow cytometric analysis showed myeloid marker CD11b expressed in the about 60% cells. The growth peak of differentiated cells was at 14 days of culture and decreased at about 33 days. No abnormality was found in the karyotype analysis of expanded cells. No tumor was found in the nude mice injected with expanded cells after 35 days and the expanded cells had the ability of phagocytizing bacteria. It is concluded that the cells, differentiated from CD34(+) cells, expanded ex vivo possess the function of granulocyte and it was safe for clinical trial.
Animals ; Antigens, CD34 ; analysis ; Cell Differentiation ; Fetal Blood ; cytology ; Granulocytes ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; Humans ; Karyotyping ; Mice ; Mice, Inbred BALB C ; Phagocytosis

OBJECTIVETo explore the effect of Modified Sijunzi Decoction (MSD) on the bone metabolism of prednisone intervened adriamycin-induced nephropathy rats.

METHODSThe adriamycin-induced nephropathy rat model was prepared. Totally 50 SD rats were randomly divide into five groups, i.e., the model group, the hormone group, the Chinese medicine (CM) group, the CM + hormone group, and the normal control group. The 24-h urine samples were collected on the 7th, 21st, and 35th day after modeling. The 24-h urine protein was measured by biuret colorimetry. Serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), osteocalcin (BGP), and tartrate-resistant acid phosphatase (TRACP) were determined by ELISA. Expressions of OPG and RANKL in the tibia tissue were detected using real-time quantitative PCR and Western blot.

RESULTS(1) Compared with the normal control group, the 24-h urine protein increased in each group on the 7th, 21st, and 35th day (P < 0.05, P < 0.01). Compared with the model group, the 24-h urinary protein decreased in the hormone group and the CM + hormone group (P < 0.05, P < 0.01). The decrement was more obvious along with the treatment time went by (P < 0.05, P < 0.01). There was statistical difference in the reduction of urine protein on the 35th day between the CM group and the model group (P < 0.05). (2) Compared with the 21st-day of the same group, the serum levels of TRACP and RANKL increased (P < 0.05, P < 0.01). Compared with the model group, the serum levels of the TRACP and RANKL increased (P < 0.05, P < 0.01), OPG and BGP decreased (P < 0.05, P < 0.01) in the hormone group. Compared with the CM group at the same period, serum OPG level decreased and the RANKL level increased in the hormone group and the CM + hormone group (P < 0.05, P < 0.01). Besides, the serum level of TRACP increased and BGP decreased (P < 0.05, P < 0.01). Compared with the hormone group at the same period, OPG and BGP increased (P < 0.05, P < 0.01), RANKL decreased (P < 0.01) in the CM + hormone group. On the 35th day TRACP decreased (P < 0.01). (3) Compared with the normal group, mRNA expressions of OPG and RANKL on the 21st day increased (P < 0.05, P < 0.01), mRNA expressions of OPG and RANKL on the 35th day decreased in the model group (P < 0.01). Compared with the CM group at the same period, OPG mRNA expression decreased (P < 0.01) and RANKL mRNA expression increased in the hormone group (P < 0.05). OPG mRNA expression decreased in the CM +hormone group (P < 0.05). (4) Compared with the hormone group on the 21st day, the OPG level decreased and the RANKL protein increased (both P < 0.05). RANKL decreased in the CM + hormone group (P < 0.05). Compared with the model group at the same period, OPG decreased and RANKL increased in the hormone group (P < 0.01). Compared with the CM group at the same period, OPG decreased (P < 0.01), RANKL increased (P < 0.01) in the hormone group and the CM + hormone group. Compared with the hormone group at the same period, OPG increased and RANKL decreased in the CM + hormone group (both P < 0.01).

CONCLUSIONSPrednisone could induce osteoporosis through the OPG/RANKL/RANK pathway. MSZ could slow down the formation of prednisone-induced osteoporosis through promoting osteoblast differentiation, and inhibiting osteoclastogenesis.

Acid Phosphatase ; metabolism ; Animals ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; Isoenzymes ; metabolism ; Male ; Nephrosis ; chemically induced ; metabolism ; Osteocalcin ; metabolism ; Osteoprotegerin ; metabolism ; Prednisone ; pharmacology ; RANK Ligand ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tartrate-Resistant Acid Phosphatase ; Tibia ; metabolism

Objective To explore the performance of mobile health platform for standardized management of pregnant women with gestational diabetes mellitus(GDM). Methods A randomized controlled trial was conducted,in which 295 women with GDM were randomized into two groups(traditional management group and mobile health management group)by a computer-generated sequence.The traditional management group accepted standardized GDM management,and the mobile health management group was supplemented by mobile health management based on the standardized management.The glycemic control rate and the incidences of low birth weight,macrosomia,preterm birth,premature rupture of membranes,postpartum hemorrhage after cesarean section,neonatal asphyxia,malformation,and admission to the neonatal intensive care unit were compared between the two groups. Results The glycemic control rate in mobile health management group was significantly higher than that in the traditional management group [(67.22±22.76)%
Cesarean Section ; Diabetes, Gestational/therapy* ; Female ; Fetal Macrosomia ; Humans ; Infant, Newborn ; Pregnancy ; Pregnancy Outcome ; Premature Birth ; Telemedicine

To investigate the effects of exogenous NaHS on myelin basic protein (MBP) and learning and memory of hippocampal neurons in mice with spinocerebellar ataxia type 3 (SCA3) and its therapeutic significance. Twelve male normal mice were randomly selected as normal control group (NC Group), and 48 SCA3 mice were randomly selected as SCA3 model group (M Group), low dose group (NL Group, 10 μmol/kg), medium dose group (NM Group, 50μmol/kg) and high dose group (NH Group, 100 μmol/kg), 12 rats in each group. The drug treated groups were injected with NaHS intraperitoneally once a day for 4 weeks. The changes of learning and memory ability of SCA3 mice before and after the intervention of different doses of NaHS were determined by Morris water maze, the content of hydrogen sulfide (HS) in hippocampus was measured by spectrophotometry, the expression of MBP was detected by immunohistochemistry, and the morphological changes of neuron myelin sheath were observed by electron microscope. Compared with the control group, the learning and memory ability of SCA3 mice was decreased significantly (P＜0.05), and the content of HS in hippocampus was decreased (P＜0.05). After different doses of exogenous NaHS treatment, the learning and memory ability was improved in different degrees (P＜0.05), and the contents of HS and MBP in hippocampus of SCA3 mice were also improved in different degrees (P＜0.05). Exogenous NaHS may increase the contents of HS and MBP in the hippocampus of SCA3 mice, which may have a protective effect on the neurons, and then improve the learning and memory ability of SCA3 mice, and provide a new idea for the treatment of SCA3.

Background: Advanced technology has become a valuable tool in etiological studies of intellectual disability/global developmental delay (ID/GDD). The present study investigated the role of genetic analysis to confirm the etiology in ID/GDD patients where the cause of the disease was uncertain in central China. Methods: We evaluated 1051 ID/GDD children aged 6 months to 18 years from March 2009 to April 2017. Data concerning basic clinical manifestations were collected, and the method of etiology confirmation was recorded. Genome-wide copy number variations (CNVs) detection and high-throughput sequencing of exons in the targeted regions was performed to identify genetically-based etiologies. We compared the incidence of different methods used to confirm ID/GDD etiology among groups with differing degrees of ID/GDD using the Chi-square or Fisher exact probability test. Results: We recruited 1051 children with mild (367, 34.9%), moderate (301, 28.6%), severe (310, 29.5%), and profoundly severe (73, 6.9%) ID/GDD. The main causes of ID/GDD in the children assessed were perinatal factors, such as acquired brain injury, as well as single gene imbalance and chromosomal gene mutation. We identified karyotype and/or CNVs variation in 46/96 (47.9%) of cases in severe ID/GDD patients, which was significantly higher than those with mild and moderate ID/GDD of 34/96 (35.4%) and 15/96 (15.6%), respectively. A total of 331/536 (61.8%) patients with clear etiology have undergone genetic analysis while 262/515 (50.9%) patients with unclear etiology have undergone genetic analysis (χ² = 12.645, P < 0.001). Gene structure variation via karyotype analysis and CNV detection increased the proportion of children with confirmed etiology from 51.0% to 56.3%, and second-generation high-throughput sequencing dramatically increased this to 78.9%. Ten novel mutations were detected, recessive mutations in X-linked genes (ATPase copper transporting alpha and bromodomain and WD repeat domain containing 3) and dominant de novo heterozygous mutations in X-linked genes (cyclin-dependent kinase like 5, protocadherin 19, IQ motif and Sec7 domain 2, and methyl-CpG binding protein 2) were reported in the study. Conclusions The present study indicates that genetic analysis is an effective method to increase the proportion of confirmed etiology in ID/GDD children and is highly recommended, especially in ID/GDD children with uncertain etiology.