BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) have been widely accepted by medical investigators due to their advantages including easy obtaining, minimal invasion, with infinite proliferation and multi-differential potential, and without immunological rejection in the autologous transplantation. OBJECTIVE: The goal of this study is to isolate and purify rat bone marrow MSCs in vitro, so as to observe the effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow MSCs.DESIGN: A randomized controlled animal experiment.SETTING: Hip Center, First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.MATERIALS: Forty healthy male SD rats of SPF grade, weighing 170 to 180 g, were provided by the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine. The protocol was performed in accordance with ethics guidelines for the use and care of animals. The involved rats were divided into 4 groups by random digit table with 10 rats in each: normal control group, high-, middle-, and low-concentration groups. METHODS: This study was performed at the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine between January and March 2005. Rat bone marrow MSCs were isolated and purified by Percoll density gradient centrifugation, and cultured in vitro to establish rat bone marrow MSCs culture system. Rats in the high-, middle-, and low-concentration groups were intragastrically administrated with 4.4, 2.2 and 1.1 g/kg serum containing kidney-tonifying and blood-activating Chinese herbs, which equaled to 20, 10 and 5 times of adult dosage, respectively. Rats in the normal control group were intragastrically administrated with purified water for 1 week. One hour after the last administration, 6 mL blood was taken from abdominal aorta of each rat under the aseptic condition. Then, it was centrifuged at 2 000 r/min for 15 minutes, and meanwhile serum was collected. 10% rat serum containing kidney-tonifying and blood-activating Chinese herbs was added to the medium in the high-, middle-, and low-concentration groups, while 10% fetal bovine serum was added in the normal control group. MAIN OUTCOME MEASURES: ① MSCs growth status; ② MSCs morphology was observed by HE staining and Giemsa's staining; ③ MSCs antigen expression was detected by an immunocytochemical method; ④ Effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on MSCs growth.RESULTS: ①The primarily cultured bone marrow MSCs began to adhere to the wall 24 hours later and 80% of them reached the confluence 7 days later. ② MSCs took appearance in long shuttle shape or polygon. These cells were little. Nuclei were located in the middle part of cells or a little deviation. The ratio of nucleus to cytoplasm was a little high. ③CD44 expression was found in the cytoplasm of mononuclear cells, and colored blue. Partial MSCs expressed c-Kit. Their cytomorphology and phenotypic expression have the characteristics of MSCs. ④Three days after serum containing kidney-tonifying and blood-activating Chinese herbal medicine being added to high-, middle-, and low-concentration groups, the number of bone marrow MSCs was dose-dependently increased as compared with that in the normal control group. CONCLUSION: Serum containing kidney-tonifying and blood-activating Chinese herbs promotes the in vitro proliferation of bone marrow MSCs.

OBJECTIVETo investigate the effect of reverse saphenous nerve neurocutaneous flaps for skin defects of forefoot.

METHODSIn the anatomic study, 50 cadaveric feet were injected with red latex and the anastomosis, distribution and external diameters of medialtarsal artery, medial anterior malleolus artery, medial plantar artery, the superficial branch of the medial basal hallucal artery and saphenousnerve nutritional vessels were observed. Based on anatomic research results, we designed the reverse saphenous nerve neurocutaneous flaps for repairing skin defects of forefoot.

RESULTSThe blood supply of reverse saphenous nerve neurocutaneous flaps were based on the vasoganglion, which consist of arterial arch at the superior border of abductor hallucis and arterial network on the surface of abductor hallucis around the saphenous nerve and medial pedis flap. From Oct. 2006 to Oct. 2011, the reverse saphenous nerve neurocutaneous flaps were used to repair skin defects of forefoot in 11 cases. The flap size ranged from 2.5 cm x 3.5 cm to 7.5 cm x 8.5 cm. The wounds at donor site were covered with full-thickness skin graft. All flaps survived completely with no ulcer at the donor site. 11 cases were followed up for 6 to 18 months( mean, 10 months). The skin color and texture were satisfactory. The patients could walk very well.

CONCLUSIONSIt is reliable to repair the skin defects of forefoot with reverse saphenous nerve neurocutaneous flaps. It is easily performed with less morbidity. This flap should be considered as a preferential way to reconstruct skin defects of forefoot.

Arteries ; anatomy & histology ; Cadaver ; Female ; Foot ; blood supply ; innervation ; Forefoot, Human ; injuries ; surgery ; Humans ; Male ; Muscle, Skeletal ; anatomy & histology ; Reconstructive Surgical Procedures ; Skin Transplantation ; methods ; Surgical Flaps ; blood supply ; innervation ; Transplant Donor Site ; surgery

Objective To investigate the role of hypoxia induced factor-1 α (HIF-1 α) signaling pathway in development of nonacoholic fatty liver disease (NAFLD) by using a rat model.Methods Forty SD rats were randomly divided into 5 groups (normal control group,4 weeks model group,8 weeks model group,12 weeks model group,and 16 weeks model group according to random number table).NAFLD model was induced by high fat diet.Serum biochemical parameters and liver histological examinations were observed.The HIF-1α,peroxisome proliferator-activated receptor α (PPARα),and nuclear factor-κB (NF-κB) mRNA transcriptions of liver tissue were detected with realtime PCR.Results Compared with the control group,a remarkable rise of serum alanine amiotransferase,triglyceride,cholesterol,and reduction of high density lipoprotein-cholesterol were observed in rats treated with high fat diet after 12 weeks and 16 weeks (P＜0.05).Compared with model group,steatosis,grade of inflammation,and degree of fibrosis in the liver were also significantly aggravated.Fed with high fat diet for 8 weeks,the expression of HIF-1α was significantly higher than that in the control group;the expression of PPARα in 4 weeks group was significantly higher than that in the control group.The expression of PPARα was again significantly elevated in 16 weeks group (P＜0.05);NF-κB expression was increased gradually with high fat diet (P＜0.05).The expression of HIF-1 α mRNA was related with PPARα (r =0.82,P＜0.05) and NF-κB (r =0.67,P＜0.05),and PPARα mRNA was related with NF-κB (r =0.78,P＜0.05).Conclusion HIF-1 α signaling may regulate PPARα/NF-κB and seems to be related with development of NAFLD.

Objective To explore the roles of p53 in ionizing radiation induced MCF-7 cell cycle uncoupling. Methods The p53 knock-down models was established in MCF-7 with retrovirus packaged particles from 293T cells through calcium acid phosphate co-precipitation, then Western blot was used to detect the protein expression. Flow cytometry(FCM) was used to analyze the cell cycle uncoupling and polyploid after irradiation. Results Compared with p53+/+ group, the percentages of G0/G1 cells in p53 -/- group decreased, while those of S and G2+M increased (P < 0.01). In polyploidy analysis 2N cells decreased, whereas both 4N and 8N cells =increased (P<0.01). Compared with sham-irradiation, 4 Gy X-ray led to the decrease of G0/G1, S cells, and the increase of G2+M cells. The increase of 2N cells and decrease of 4N and 8N cells were observed in both p53+/+ and p53-/- cells. Compared with p53+/+ +IR group, the decrease of G0/G1 and S cells and the increase of C2 + M cells were significant (P < 0.01) in p53-/-+ IR groups. 2N cells decreased, 4N cells increased, but no changes in 8 N cells occurred. Conclusion Radiation might induce G2 arrest and cycle uncoupling, p53 plays a role in the regulation of G2 arrest, but no role in cycle uncoupling.

The aim of the present study was to investigate the effects and mechanisms of quercetin on plantar incision-induced matrix metalloproteinase (MMP)-9 and MMP-2 activity,microglia activation and the analgesic effect of quercetin on plantar incision surgery-treated mice.Postoperative pain model was mediated by plantar incision surgery and Von Frey Hairs was used to test the mechanical pain threshold.The activity of MMP-9 and MMP-2 in spinal cord was evaluated by gelatin zymography.The marker of microglia ionized calcium binding adapter molecule 1 (IBA-1),phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) was detected by Western blot.Results showed that quercetin (20,40,80 mg/kg,ip) significantly inhibited plantar incisioninduced mechanical allodynia and suppressed the activity of MMP-9 and MMP-2 in the spinal cord.Moreover,quercetin also markedly inhibited plantar incision-induced up-regulation of IBA-1 and p-p38 in spinal cord.In conclusion,quercetin may alleviate postoperative pain by suppressing MMP-9 and MMP-2 activity in microglia.

Objective: To observe the effect of electroacupuncture (EA) at Zusanli (ST 36) on the gastrointestinal motility and the ultrastructures of pacemaker cells [the interstitial cells of Cajal (ICC)] in diabetic gastroparesis (DGP) rats and explore the mechanism of EA for DGP.Methods: A total of 50 Sprague-Dawley (SD) rats were randomly divided into group A, group B, group C, group D and group E, with 10 rats in each group. Group A was the blank control; a single intraperitoneal injection of 2% streptozotocin (STZ) was performed in rats of group B, group C, group D and group E, with high glucose and high fat diet for 8 weeks to establish the DGP rat models. Group B was the model group and the rats did not receive any treatment; group C was EA at acupoint group and the rats received EA at Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6); group D was EA at non-acupoint group and the rats received EA at the control points of Zusanli (ST 36), Liangmen (ST 21) and Sanyinjiao (SP 6); group E was metoclopramide group and the rats were treated by intragastric administration of metoclopramide. Blood glucose was detected using ONE TOUCH blood glucose meter; gastric emptying rate and small intestine migration rate were measured using intragastric phenol red; ultrastructures of gastric antrum ICC were detected by transmission electron microscopy.Results: The differences of blood glucose between group B, group C, group D, group E versus that of group A were statistically significant after modeling (P<0.01); after treatment, the differences of blood glucose between group D, group E versus that of group C were statistically significant (P<0.05,P<0.01); the gastric emptying rate of rats in group B was statistically significant different from that in group A (P<0.01); the gastric emptying rate of rats in group C was statistically significant different from that in group B (P<0.01). The migration rates of rats' small intestines in group B, group C, group D and group E were all statistically significant different from that in group A (P<0.01); the migration rate of rats' small intestines in group C was statistically significant different from that in group B (P<0.01). The ultrastructure of rat's ICC in group B showed apoptosis compared with that in group A; rat's ICC in group C had complete basement membrane, more cytoplasm mitochondria, Golgi and rough endoplasmic reticulum, showing clear structure, occasional mitochondria swelling and gap junctions with adjacent smooth muscle cells; there were no significant differences between group D, group E versus group B.Conclusion: EA at Zusanli (ST 36) plus other acupoints can regulate the blood glucose and promote gastrointestinal motility in DGP rats, and the mechanism may be related to repairing the damaged ICC structure.

Objective To evaluate the role of spinal c?Jun N?terminal kinase ( JNK ) signaling pathway in incisional pain in rats. Methods Sixty?three adult male Sprague?Dawley rats, weighing 200-250 g, were randomly divided into 3 groups ( n=21 each) using a random number table: incisional pain group ( IP group) , dimethyl sulfoxide ( DMSO) group, and JNK inhibitor SP600125 group ( SP group) . A 1?cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the hindpaw in anesthetized rats. In group DMSO, 10% DMSO 10 μl was injected intrathecally at 30 min before surgery. In group SP, SP600125 25 μg (in 10 μl of 10% DMSO) was injected intrathecally at 30 min before sur?gery. Six rats in each group were sacrificed, and the mechanical paw withdrawal threshold ( MWT) and thermal paw withdrawal latency ( TWL) were measured at 24 h before establishment of the model and 2, 6, 24, 48 and 72 h after establishment of the model. After measurement of the pain threshold at 24 h before establishment of the model and 6, 24, 48 and 72 h after establishment of the model, the lumbar segment of the spinal cord was removed for determination of the expression of phosphorylated JNK ( p?JNK) by im?munofluorescence. Results The MWT was significantly lower, the TWL was shorter, and the expression of p?JNK was lower at each time point after establishment of the model than at 24 h before establishment of the model in group IP (P<0?05). Compared with group IP, the MWT was significantly increased, the TWL was prolonged, and the expression of p?JNK was down?regulated at each time point after establishment of the model in group SP ( P<0?05) , and no significant change was found in the parameters mentioned a?bove in group DMSO ( P>0?05) . Conclusion Spinal JNK signaling pathway is involved in the develop?ment and maintenance of incisional pain in rats.

OBJECTIVEThis aimed to investigate the effect of specific sequence oligodeoxynucleotide MT01 on the biological properties of osteoblasts invaded by Porphyromonas gingivalis (P. gingivalis ) by evaluating proliferation, cell cycle, and apoptosis.

METHODSMG63 osteoblasts were recovered and incubated with MT01, CpG ODN, metronidazole (MNZ), and gentamicin (GEN) for 3 h. P. gingivalis (the multiplicity of infection was 100:1) was added subsequently and cocultured for another 24 and 48 h. Cells with PBS comprised the blank group, whereas cells with P. gingivalis comprised the negative controls. Six experimental groups were established: PBS group, P. gingivalis group, MT01+P. gingivalis group, CpG ODN+ P. gingivalis group, MNZ+P. gingivalis group, and GEN+P. gingivalis group. The proliferative ability was measured by methyl thiazolyl tetrazolium assay, and the percentages of apoptosis and cell cycle were examined by flow cytometry.

RESULTSCompared with the blank group, proliferation increased significantly in the MT01+P. gingivalis group (P < 0.05). The ratio of cells was lower at the G₁ phase and higher at the S phase in the MT01+P. gingivalis group compared with the results in the P. gingivalis group (P < 0.05). Early cell apoptosis in the MT01+P. gingivalis group was significantly lower than that in the P. gingivalis group (P < 0.05).

CONCLUSIONMT01 can promote the proliferation, reduce the ratio of the G₁phase, increase the ratio of the S phase, and inhibit the early apoptosis of osteoblasts invaded by P. gingivalis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Division ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gentamicins ; pharmacology ; Humans ; Metronidazole ; pharmacology ; Oligodeoxyribonucleotides ; pharmacology ; Osteoblasts ; cytology ; drug effects ; Porphyromonas gingivalis ; pathogenicity

Objective:To explore clinical significance of sinus heart rate turbulence (HRT) phenomenon in aged pa‐tients with stable angina pectoris (SAP) .Methods :A total of 120 aged SAP patients ,who received 24h DCG in our hospital from Jan 2013 to Oct 2015 ,were selected as SAP group .Meanwhile ,another 144 aged patients ,who re‐ceived 24h DCG examination simultaneously and coronary angiography results were normal ,were regarded as nor‐mal control group .According to coronary lesion severity ,SAP group was further divided into single vessel coronary disease group (single vessel group ,n=35) ,double‐vessel coronary disease group (double‐vessel group ,n=48) and multi‐vessel coronary disease group (multi‐vessel group ,n=37) .The 24h DCG ,HRT indexes ,including turbulence onset (TO) and turbulence slope (TS) ,were measured and compared among all groups .Results:Compared with normal control group ,there was significant rise in TO [(0.77 ± 0.37)% vs .(1.26 ± 0.92)% ] and significant reduc‐tion in TS [(5.45 ± 4.02) ms/RR interval vs .(1.53 ± 0.70) ms/RR interval] ,P<0.01 both ;significant rise in ab‐normal rates of TO (19.44% vs .42.50% ) ,TS (15.97% vs .31.67% ) and TO + TS (11.11% vs .30.83% ) in SAP group ,P<0.01 all .Compared with single vessel group ,there was significant rise in TO [(0.66 ± 0.22)% vs .(1.28 ± 1.11)% vs .(1.46 ± 1.20)% ] and significant reduction in TS [ (2.04 ± 0.82) ms/RR interval vs .(1.66 ± 0.38) ms/RR interval vs .(1.29 ± 0.58) ms/RR interval] in double‐vessel group and multi‐vessel group ,and TO of multi‐vessel group was significantly higher than that of double‐vessel group ,TS of multi‐vessel group was significantly low‐er than that of double‐vessel group , P<0.01 all .Conclusion:Sinus heart rate turbulence can be used as risk predic‐tor for aged patients with stable angina pectoris ,which can provide basis for clinical effective treatment and progno‐sis assessment .

Objective To investigate the effect of retrograde sartorius myocutaneous flap for reparing skin defects of leg.Methods In the anatomic study,50 cadaveric lower limb were injected with red latex and the origin,diameter,course,distribution and anastomosis of sartorius's arteries were observed.Arteriographies were made in 4 sides of fresh specimens to study the arterial anastomosis in sartorius.Based on anatomic research results,we designed the retrograde sartorius myocutaneous flap for reparing skin defects of leg.Results Nutrient arteries of sartorius represented segnental distribution,Link-pattern arterial anastomosises were formed in sartorius by branches of adjacent vascular pedicles.Cutaneous arteries and musculocutaneous arteries above deep fascia formed interlocking arterial anastomosises net which provided blood supply for the skin on sartorius.The arterial branches in the distal 2/5 of sartorius came from saphenous artery,composed an arterial network around knee joint,which consist of anatomic basis for the blood supply of retrograde sartorius myocutaneous flap.Form February,2010 to April,2014,the retrograde sartorius myocutaneous flap were used to repair skin defects of leg in 2 cases.The flap size ranged from 7 cn×18 cm to 12 cm×25 cm.All flaps survived successfully with no ulcer.2 cases were followed up for 7 to 16 months.The skin color and texture were satisfactory.Conclusion the retrograde sartorius myocutaneous flap has constantly,reliable blood supply,and easily performed.It is an effective method for the reconstruction skin defects of leg.