Objective The aim of this study was to explore the correlation between the methylation status of microRNA let-7a-3 in esophageal squamous cell carcinoma(ESCC)and the expression of insulin-like growth factor 2(IGF-Ⅱ).Methods The methylation specific PCR(qMSP)was used to detect the methylation status of let-7a-3 in 83 cases of esophageal cancer and corre-sponding adjacent normal tissues.The enzyme linked immunosorbent assay(ELISA)was used to detect the expression of IGF-Ⅱ in plasma.Results The degree of let-7a-3 methylation in cancer tissues of 83 patients with ESCC was significantly higher than that in normal tissues adjacent to cancer(P<0.001).The expression of IGF-Ⅱ in the plasma of 83 patients with ESCC was positively corre-lated with the methylation degree of let-7a-3,which was statistically significant(r=0.600,P<0.001).Conclusion microRNAlet-7a-3 may participate in the occurrence and progression of ESCC by regulating the methylation of downstream molecules,which is of great significance for understanding the mechanisms of ESCC development and providing a basis for the diagnosis and prognosis of ESCC.

BACKGROUND: Insufficient angiogenesis and the lack of skin appendages are critical challenges in cutaneous wound healing. Stem cell-fabricated cell sheets have become a promising strategy, but cell sheets constructed by a single cell type are inadequate to provide a comprehensive proregenerative microenvironment for wound tissue. METHODS: Based on the communication between cells, in this study, bone marrow mesenchymal stem cells (BMSCs) and hair follicle stem cells (HFSCs) were cocultured to fabricate a composite cell sheet (H/M–CS) for the treatment of fullthickness skin wounds in mice. RESULTS: Experiments confirmed that there is cell–cell communication between BMSCs and HFSCs, which enhances the cell proliferation and migration abilities of both cell types. Cell–cell talk also upregulates the gene expression of proangiogenic-related cytokines in BMSCs and pro-hair follicle-related cytokines in HFSCs, as well as causing changes in the properties of secreted extracellular matrix components. CONCLUSIONS Therefore, the composite cell sheet is more conducive for cutaneous wound healing and promoting the regeneration of blood vessels and hair follicles.

Objective: To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and G protein-coupled receptor kinase 2 (GRK2) in the development of persistent postoperative pain in rats. Methods: Pathogen-free healthy male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were used in this study.Sixty rats in which intrathecal catheters were successfully implanted were divided into 6 groups (n=10 each) using a random number table method: sham operation group (group S), sham operation plus dimethyl sulfoxide (DMSO) group (group D), sham operation plus GRK2 degradation inhibitor MDL28170 group (group M), persistent postoperative pain group (group PPP), persistent postoperative pain plus DMSO group (group PPP+ D) and persistent postoperative pain plus MDL28170 group (group PPP+ M). Persistent postoperative pain was evoked by skin/muscle incision and retraction (SMIR). Immediately after operation and at 1, 2 and 3 days after operation, normal saline 20 μl was intrathecally injected once a day in S and PPP groups, 5% DMSO 10 μl was intrathecally injected once a day in D and PPP+ D groups, and MDL28170 10 μl (50 μg) was intrathecally injected once a day in M and PPP+ M groups.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation and 3, 7, 14 and 21 days after operation (T_1-4). Four rats in each group were selected after behavioral testing at T₃ and sacrificed, and the L_4-6 segments of the spinal cord were removed for determination of the expression of phosphorylated p38MAPK (p-p38MAPK) by Western blot. Results: There was no significant difference in MWT at each time point or expression of p-p38MAPK among group S, group D and group M (P>0.05). Compared with group S, the MWT was significantly decreased, and the expression of p-p38MAPK was up-regulated in PPP, PPP+ D and PPP+ M groups (P<0.05). Compared with group PPP, the MWT was significantly increased, and the expression of p-p38MAPK was down-regulated in group PPP+ M (P<0.05), and no significant change was found in the MWT or expression of p-p38MAPK in group PPP+ D (P>0.05). Conclusion Down-regulated expression of spinal GRK2 can promote the activation of p38MAPK in the spinal cord and is involved in the development of persistent postoperative pain in rats.

Objective To investigate the effects of inverse ratio ventilation on pulmonary function and ce-rebral oxygen saturation in elderly patients with single lung ventilation.Methods Sixty patients scheduled for elec-tive radical resection of esophageal cancer were divided into 2 groups(n=30 for each group)using a random num-ber table:the experiment group(group A)and the control group(group B). During the two lung ventilation,the ventilator parameters were set as tidal volume(VT)7 mL/kg,inspiratory to expiratory ratio 1:2. During one lung ventilation,the I:E ratio was 1.5:1 in the group A and 1:2 in the group B. At 15 min after two lung ventilation (T1),20 min after one lung ventilation(T2),60 min after one lung ventilation(T3)and 15 min after restarting two-lung ventilation(T4),the blood gas analysis was measured and recorded for the hemodynamics,respiratory me-chanics index and cerebral oxygen saturation respectively. Results Compared with the B group,the Ppeak and VD/VT at T2~T4in the group A were lower while PaO2,Pmean and Cdyn were higher(P<0.05).During one lung ventilation,the incidence of rSO2< 50% or rSO2decreased more than 20% in the group A was lower than that in the group B(P<0.05).The PaO2,Cdyn and rSO2of the two groups at T2~T4were significantly lower and Ppeak, Pmean,PaCO2,VD/VT were significantly higher than the baseline(T1)(P < 0.05). Conclusion During one-lung ventilation,prolonged inspiratory time can improve pulmonary function and lung compliance without increas-ing peak airway pressure,reduce the decline of rSO2 at the same time.

Objective To explore the effect of high-dose methylprednisolone combined with femoral nerve block on postoperative analgesia and recovery following total knee arthroplasty(TKA).Methods We performed a randomized,placebo-controlled,double-blind study. 60 patients were divided into a study group(groupMF)and a control group(F group).Patients in group MF received high-dose methylprednisolone combined with femoral nerve block. Patients in group F received equivalent saline combined with femoral nerve block. The rest pain and activi-ties pain,uses of analgesic medication,24 h CRP,time to ambulation,and adverse reactions after operation were recorded.Results There were significant differences in the rest pain and activities pain at hours 6,12,24,and 48,uses of analgesic medication,24 h CRP,time to ambulation and PONV(P < 0.05),group MF was better than group F.There was no significant difference in other adverse reactions(P>0.05).Conclusions High-dose methylprednisolone combined with femoral nerve block can effectively relieve pain and reduce uses of analgesic drugs after TKA,with less systemic inflammatory response and PONV.It is helpful for the early recovery in knee function.

Objective To investigate effects of preoperative application of parecoxib on postoperative analgesia and coagulation function in neurosurgical patients.Methods A total of 90 patients (38 males and 52 females,ASA physical status Ⅰ or Ⅱ) undergoing crainotomy were randomly divided into two groups(n=45): parecoxib group (group P) and control group (group C).At 30 min before operation,group P received intravenous injection of parecoxib 40 mg (5 ml),group C intravenous injection of saline 5 ml.Postoperative patient-controlled intravenous analgesia (PCIA) was performed in all patients.PCIA formula of sufentanil 2 μg/kg+tropisetron 0.2 mg/kg,were diluted with normal saline to 120 ml.The visual analogue scale (VAS),the total and effective PCIA pump compressions,Ramsay sedation scale of 2,4,16,24,48 h after operation were recorded.Coagulation function was measured before and 2 h,48 h after parecoxib administration.Meanwhile,adverse reactions were recorded.Results Comparion of VAS between the two groups was made within 48 h after surgery,the total and effective PCIA pump compressions,were much more in group C than in group P (P<0.05).Ramsay sedation scale of group C was higher than that in group P at 2 h after operation.There were no significant differences in coagulation function.And the percentage of patients′ adverse effects in group P was lower than that in group C (P<0.05).Conclusion Parecoxib,as an analgesic,can enhance analgesic effect of sufentanil PCIA.Not only does it reduce the amount of sufentanil and incidence of adverse reactions,but also it has no significant effect on blood coagulation function.

Objective To evaluate the role of sonic hedgehog (SHH) signaling pathway in spinal neurons in morphine tolerance (MT) in mice.Methods Pathogen-free healthy female Kunming mice,weighing 20-25 g,aged 8-10 weeks,were used in the study.MT was induced with morphine 10 mg/kg injected subcutaneously twice a day for 7 consecutive days.The experiment was performed in two parts.Experiment Ⅰ Forty-eight mice were randomly assigned into 2 groups:control group (group C,n =8) and MT group (group M,n=40).The thermal pain threshold (TPT) was measured at 1 day before morphine injection and 1,3,5,7 and 14 days after the end of injection.Eight mice in each group were sacrificed at 2 h after measurement of TPT at each time point after the end of injection in group M or at 2 h after the last measurement of TPT in group C,and the lumbar segment (L4-6) of the spinal cord was removed.Experiment Ⅱ Forty-eight mice were randomly assigned into 6 groups (n=8 each):SHH inhibitor cyclopamine plus MT group (group CP+M),cyclopamine solvent plus MT group (group D1 +M),SHH agonist SAG plus MT group (group SAG+M),SAG solvent plus MT group (group D2+M),MT plus cyclopamine group (group M+CP) and morphine plus cyelopamine solvent group (group M+D1).At 15 min before morphine injection,cyclopamine 10 mg/kg was injected subcutaneously in group CP+M,and SAG 5 mg/kg was injected subcutaneously in group SAG+M.Cyclopamine 10 mg/kg was injected subcutaneously once a day during the 1-3 days after the end of morphine injection in group M+CP.The TPT was measured before injection of morphine,at 30 min after the first injection of morphine every day and at 1-3 days after the end of morphine injection.The animals were sacrificed at 2 h after the last measurement of TPT,and the lumbar segment (L4-6) of the spinal cord was removed for determination of the expression of SHH signaling pathway-related proteins SHH,ptch1,smo,gli1 and gli3 using Western blot.Results Experiment Ⅰ Compared with group C,the TPT was significantly decreased at 1 and 3 days after the end of morphine injection (P＜0.05),no significant change was found in TPT at 5-14 days after the end of morphine injection (P＞0.05),and the expression of SHH,smo and glil at 1-5 days after the end of morphine injection,of ptchl at 1 and 3 days after the end of morphine injection and of gli3 at 7 days after the end of morphine injection was up-regulated in group M (P＜0.05).Experiment Ⅱ Compared with group D1+M,the TPT was significantly increased,the expression of SHH,ptchl,smo and glil was down-regulated,and gli3 expression was up-regulated in group C P+M (P＜0.05).Compared with group D2+M,the TPT was significantly decreased,the expression of SHH,ptch1,smo and glil was up-regulated,and gli3 expression was down-regulated in group SAG+M (P＜0.05).There was no significant difference in the parameters mentioned above between group M+CP and group M+D1 (P＞0.05).The TPT was significantly lower on 3rd-7th days after beginning of morphine injection and 1-3 days after the end of morphine injection than at 30 min after the first injection of morphine in group CP+M (P＜0.05).Conclusion The mechanism underlying the development of MT is partially related to activation of SHH signaling pathway in spinal neurons of mice,however,the maintenance mechanism has no marked relationship with it.

Objective To evaluate the effects of intrathecal low-dose naloxone,morphine and fentanyl on the expression of motillin (MTL) in the hippocampus of rats with incisional pain.Methods Seventy-two healthy male Sprague-Dawley rats,weighing 180-220 g,aged 6-8 weeks,in which intrathecal catheters were successfully implanted,were randomly divided into 6 groups (n =12 each) using a random number table:normal saline group (NS group),incisional pain group (P group),morphine + fentanyl + incisional pain group (MFP group),and naloxone (0.2,1.0 and 5.0 ng/kg) + morphine + fentanyl groups (MFPN1,MFPN2 and MFPN3 groups).Incisional pain was induced by an incision made into the plantar surface of the right hindpaw.At 20 min before induction of incisional pain,the mixture of morphine 5 μg/kg and fentanyl 0.25 mg/kg was injected intrathecally in group MFP,and the mixture of naloxone 0.2,1.0 and 5.0 ng/kg,morphine and fentanyl were injected intrathecally in MFPN1,MFPN2 and MF-PN3 groups,respectively.Six rats in each group were selected,and the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before intrathecal catheterization (T0,baseline),at 24 h before induction of incisional pain (T1),and at 1,3 and 6 h after operation (T2-4).The left 6 rats in each group were selected and sacrificed at 6 h after operation,and the hippocampi,body of the stomach and duodenum were removed for detection of MTL content by enzyme-linked immunosorbent assay.Results Compared with group NS,the MWT was significantly decreased,and the TWL was shortened at T2-4 in P and MFPN3 groups,the MWT was significantly decreased,and the TWL was shortened at T4 in group MFPN1,and the TWL was prolonged at T2 in group MFPN2,the MTL contents in hippocampus and body of the stomach were significantly decreased in P,MFP,MFPN1 and MF-PN3 groups,the MTL contents in duodenum were increased in P and MFPN3 groups,and the MTL contents in duodenum were decreased in MFP and MFPN1 groups (P＜0.05),and no significant change was found in the parameters mentioned above in group MFPN2 (P＞0.05).Conclusion Intrathecal naloxone 1.0 ng/kg combined with morphine and fentanyl can inhibit up-regulation of the expression of MTL in the hippocampus of rats with incisional pain,and then is involved in the maintenance of stable gastrointestinal motility.

Objective To establish a scald-induced pain model using a constant-temperature electrical scald instrument in rats.Methods Thirty-six pathogen-free male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into 4 groups (n =9 each) using a random number table: control group (group C), scald for 5 s group (group S5), scald for 10 s group (group S10), and scald for 15 s group (group S15).The rats were anesthetized with intraperitoneal chloral hydrate.In S5, S10 and S15 groups, the plantar surface of the left hindpaw of rats were exposed to a constant-temperature electrical scald instrument (85 ℃) for 5, 10 and 15 s, respectively.The plantar surface of the left hindpaw of rats was exposed to an electrical scald instrument (room temperature) for 10 s in group C.At 1 day before treatment (T0),and 1, 3, 5, 7 and 14 days after treatment (T1-5), the mechanical and thermal pain thresholds were measured.Immediately after treatment, and at 24 h after treatment, the total body condition, wound color, and shape of the margin of the wound were observed and recorded.At 24 h after treatment, 3 rats were randomly sacrificed, and the skin from the plantar surface of the left hindpaw was removed for microscopic examination.Results Compared with group C, the thermal pain threshold at T1-2, and the mechanical pain threshold at T1-3 were significantly decreased in group S5, and the thermal pain threshold,and mechanical pain threshold were significantly decreased at T1-4 in group S10 (P＜0.05).The thermal pain threshold ＞ 25 s, and the mechanical pain threshold ＞30 g at T1-5 in group S15.The swelling in foot was bovious, burn blister appeared, and the degree of damage was aggravated in group S10 compared with S5 and S15 groups.Conclusion The scald-induced pain model is successfully established using a constant-temperature electrical scald instrument in rats.

Objective To observe a new phosphodiesterase-4 inhibitor Ro 20-1724 on ketamine anesthesia-induced learning and memory impairment and cAMP/PKA-CREB-BDNF signal pathway in immature rats.Methods Twenty-four 21 day-old SD rats were randomly divided into 4 groups (n=6 each):control group (group C) ;ketamine group (group K) ;ketamine+Ro 20-1724 group(group K+Ro) and ketamine+vehicle (0.1％ ethanol) group (group K+E).Ketamine 70 mg/kg was injected intraperitoneally(IP) once a day for 7 consecutive days in groups K,K+Ro,and K+E.Ro 20-1724 0.5 mg/kg and equal volume of vehicle were injected IP at 30 min after IP ketamine each time respectively.Morris water maze was used to assess learning and memory ability after 2 days normal feeding,the escape latency and frequency of passing the platform were recorded.The animals were killed after water maze test and the cAMP,PKA,p-CREB,and BDNF protein expression in hippocampus were detected.Results Repetitive ketamine anesthesia significantly prolonged the escape latency (P＜0.01),decreased the frequency of passing the platform(P＜0.01),and down-regulated the expression of cAMP,PKA,p-CREB,and BDNF protein ((280±31) pmol/mg vs (210± 19) mol/mg,P＜0.01 ; 1.32±0.11 vs 1.13±0.12,P＜0.01 ; 2.61 ±0.22 vs 2.03 ± ±0.19,P＜0.01 ; 1.51 ±0.14 vs 1.16±0.10,P＜0.05) ; Compared with group K,Ro 20-172,significantly attenuated the escape latency time(P＜0.05,P＜0.01)and increased the frequency of passing the platform(P＜0.01),and ameliorated the expression of cAMP,PKA,p-CREB,and BDNF protein ((210± 19) pmoL/mg vs (240± 27) pmol/mg,P ＜0.05;1.13±0.12 vs 1.28±0.12,P＜0.05;2.03±0.19 vs 2.32±0.21,2.32±0.21;1.16±0.10 vs 1.37±0.11,P＜0.01).There was no difference between group K+Ro and group C,and between group K+E and group K(P＞0.05).Conclusion ketamine anesthesia-induced learning and memory impairment can be improved by Ro 20-1724,and cAMP/PKA-CREB-BDNF signal pathway in hippocampus participated in the changes.