Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

ObjectiveTo measure the perfusion parameters of liver fibrosis of dog model with 64-slice spiral CT,and compare with positive control to reflect the degree of liver fibrosis,and analyze the correlation with VEGF values.Methods Liver fibrosis was induced in dogs by intraperitoneal injection of CC14 and high fat diet.CT perfusion scan and liver biopsies were performed.The perfusion parameters were measured according to the liver fibrosis models,and the correlation with VEGF values was analyzed.ResultsThe animals in experimental group were successfully induced different degree of liver fibrosis.In normal group,the hepatic artery perfusion,portal vein perfusion and total hepatic blood flow were (28.25 ±2.19)ml/(min · 100 g),(53.53 ± 10.71)ml/(min · 100 g) and (81.78 ± 18.56) ml/(min · 100 g).The PVP and TLP values of the liver fibrosis models of 0 - 4 stages declined gradually,and it had statistical significance,while the HAP values increased.The PVP and TLP values were positive correlated with fibrosis stage.The VEGF values of the pathological models of 0 - 4 stages ascended significantly.Conclusions The dog liver fibrosis models that are similar to human hepatic fibrosis have been successfully induced by low dose CC14 and high fat diet.CT perfusion can be used to monitor the tendency of the hemodynamic in different degree of fibrosis.VEGF may play an important role during liver fibrogenesis.

Objective:To explore the role of N-cadherin and β-catenin in the formation of spinal commissural axon projection during chicken embryonic development.Methods:Fertilized eggs were cultured for three days(stage22),N-cadherin orβ-catenin inter-ference plasmid was injected into the neural tube and in vivo electroporation was performed.Three days after the electroporation, embryos were collected,fixed with 4%PFA,embeded with OCT,and cut into frozen sections.Four groups ( knockdown of N-cadherin orβ-catenin or both of them,and control) were included in this study.Immunohistochemistry method was used to analyze the protein expression result of N-cadherin or β-catenin.The changes of spinal commissural projections were observed with GFP fluorescence.Results:During chicken embryonic development,knockdown of N-cadherin inhibited the expression of β-catenin in the spinal cord.The commissural nerve fibers projecting to the contralateral side of the spinal cord was impaired after knockdown of N-cadherin or β-catenin;this phenotype was similar after knocking down both of them.Conclusion: N-cadherin is implicated in the formation of spinal commissural projection in the developing spinal cord,possibly viaβ-catenin.

Objectives To investigate whether osteoarthritis (OA) progression can be delayed by casticin in rodent models of anterior cruciate ligament transection (ACLT).Methods Eighteen 2-month-old male C57BL/6J mice were randomised into 3 even groups (n =6) subjected respectively to sham-operation,ACLT-vehicle-treatment and ACLT-casticin-treatment.The knee capsule was dissected in the sham-operation group and ACLT on the right knee was conducted in the ACLT-vehicle-treatment and ACLT-casticin-treatment groups.Intragastric administration of the same amount of Tween-80 solution was conducted for the sham-operation and ACLT-vehicle-treatment groups;Intragastric administration of casticin of 20mg/kg was conducted once per day for the ACLT-casticin-treatment group.Bone micro CT (μCT) was quantitated to detect alterations in microarchitecture of femoral condyle subchondral bone.Tartrate resistant acid phosphatase(TRAP) stain and NOX4 immunostaining were conducted to detect relative proteins and the osteoclast changes on the subchondral bone.Articular cartilage degeneration was graded using HE and safranin O-green staining and the Mankin score criteria.Results Compared with the the sham-operation group,the subchondral bone density,trabecular bone volume fraction and trabecular thickness were decreased,and the trabecular space,positive rates of TRAP stain and NOX4 immunostaining and Mankin scores were increased in the ACLT-vehicle-treatment group.All the above comparisons were statistically significant (P ＜ 0.05).Compared with ACLT-vehicle-treatment group,the subchondral bone density and trabecular bone volume fraction were increased,and the trabecular space,positive rates of TRAP stain and NOX4 immunostaining and Mankin scores were decreased in the ACLT-casticin-treatment group.All the above comparisons were statistically significant (P ＜0.05).Conclusion As casticin may attenuate early OA progression by inhibiting NOX4 activity in subchondral bone and formation of osteoclasts,it may be a new clue to preventive therapy for OA.

Objective:To investigate the effect of modified thoracic nerve block on postoperative analgesia and early recovery of breast cancer patients after modified radical mastectomy.Methods:Sixty female breast cancer patients who were scheduled to undergo modified radical mastectomy in the Second Hospital of Shanxi Medical University from March 2019 to December 2019 were selected, and the patients were all American Society of Anesthesiologists (ASA) grade Ⅰ-Ⅱ. According to the random number table method, the patients were divided into three groups: improved group [third rib approach serratus anterior plane block (SAPB)+pectoralis major muscle superficial anterior cutaneous branch of intercostal nerve block combined with general anesthesia, STG group], SAPB combined with general anesthesia group (SG group) and general anesthesia+postoperative intravenous controlled analgesia pump group (GP group), with 20 cases in each group. VAS scores at 30 min, 2 h, 4 h, 12 h and 24 h after surgery, 15-item quality of recovery scale (QoR-15) scores at 1 d before and 24 h after surgery, sleep duration on the night after surgery, intraoperative opioid dosage, the time between stop medication and awakening, postoperative flurbiprofen ester dosage, nausea, vomiting, dizziness, puncture site infection, pneumothorax, infection and other adverse reactions were recorded.Results:The VAS scores at 30 min, 2 h, 4 h, 12 h, 24 h in the STG group were lower than those in the SG group and GP group, the VAS scores at 2 h, 12 h and 24 h after surgery in the SG group were lower than those in the GP group, and the differences were statistically significant (all P < 0.05). The QoR-15 score at 24 h in the STG group was higher than that in the SG group and the GP group [(137.85±2.81) points vs. (134.80± 2.72) points, (133.80±5.16) points], and the differences were statistically significant (all P < 0.05). The sleep duration on the night after surgery in the STG group and the SG group was longer than that in the GP group [(6.03±0.90) h, (5.48±1.12) h vs. (3.85±1.76) h], and the differences were statistically significant (all P < 0.05). The intraoperative opioid dosage and the time between stop medication and awakening in the STG group and the SG group were lower than those in the GP group, and the differences were statistically significant (all P < 0.05). The postoperative flurbiprofen ester dosage and the incidence rates of nausea, vomiting and dizziness in the STG group were lower than those in the SG group and the GP group, and the SG group was lower than the GP group, and the differences were statistically significant (all P < 0.05). Conclusion:The modified thoracic nerve block can relieve early postoperative pain, reduce the amount of intraoperative opioids, and improve the quality of patients' early recovery without increasing adverse reactions.

Objective The objective of this study was to utilize proton magnetic resonance spectroscopy (1H-MRS) to assess metabolites in cerebellar nuclei in unmedicated patients with insomnia disorder. Methods 1H-MRS was performed on cerebellar nuclei in 23 unmedicated patients with insomnia disorder (insomnia group) and 18 normal sleepers (control group). N-acetylaspartate (NAA), choline-containing compound (Cho) and creatine (Cr) were measured and the ratios of NAA/Cr and Cho/Cr were determined.Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI) were used to assess the subjective sleep quality and insomnia severity of all subjects, while State-Trait Anxiety Inventory (STAI) and Beck Depression Inventory (BDI) were used to assess the levels of anxiety and depression of all subjects. Sleep parameters of all subjects were measured by polysomnography (PSG). Results Mean NAA/Cr ratio of right cerebellar nuclei in insomnia group was significantly lower than that in control group (1.72±0.37 vs. 2.03±0.50, t=2.280, P=0.028). Mean NAA/Cr ratio of right cerebellar nuclei was significantly higher than that of left cerebellar nuclei within control group (2.03±0.50 vs. 1.68±0.21, t=3.386, P=0.004). There was no significant difference with regard to NAA/Cr ratio between bilateral cerebellar nuclei within insomnia group (t=1.416, P=0.171). Across all subjects, PSQI global scores (r=-0.369, P=0.018), and sleep latency (r=-0.437, P=0.004) and number of awakenings after sleep onset (r=-0.432, P=0.005) measured by PSG were negatively correlated with NAA/Cr ratios of right cerebellar nuclei, while percentages of stage 3 sleep (r=0.377,P=0.015) measured by PSG were positively correlated with NAA/Cr ratios of right cerebellar nuclei,respectively. Conclusion Patients with insomnia disorder have a hemispherically lateralized metabolic disturbance of NAA/Cr in right cerebellar nuclei,indicating that patients with insomnia disorder have neuronal damage in right cerebellar nuclei.

Objective: This study’s objective is to assess the efficacy and safety of Pulsed Magnetic Therapy System (PMTS) in improving insomnia disorder. Methods: Participants with insomnia disorder were randomly assigned to receive either PMTS or sham treatment for four weeks (n= 153; PMTS: 76, sham: 77). Primary outcomes are the Insomnia Severity Index (ISI) scores at week 0 (baseline), 1, 2, 3, 4 (treatment), and 5 (follow-up). Secondary outcomes are the Pittsburgh Sleep Quality Index at baseline and week 4, and weekly sleep diary-derived values for sleep latency, sleep efficiency, real sleep time, waking after sleep onset, and sleep duration. Results: The ISI scores of the PMTS group and the sham group were 7.13±0.50, 11.07±0.51 at week 4, respectively. There was a significant group×time interaction for ISI (F3.214, 485.271=24.25, p<0.001, ηp 2=0.138). Only the PMTS group experienced continuous improvement throughout the study; in contrast, the sham group only experienced a modest improvement after the first week of therapy. At the end of the treatment and one week after it, the response of the PMTS group were 69.7% (95% confidence interval [CI]: 58.6%–79.0%), 75.0% (95% CI: 64.1%–83.4%), respectively, which were higher than the response of the sham group (p<0.001). For each of the secondary outcomes, similar group×time interactions were discovered. The effects of the treatment persisted for at least a week. Conclusion PMTS is safe and effective in improving insomnia disorders.

Objective To analyze the differences in immune system between Npc1 gene mutant (Npc1-/ -) and wild-type (Npc1+/ +) mice for better understanding the pathogenesis of Niemann-Pick disease type C1 (NPC1) from an immunological perspective and providing reference for NPC1 treatment in clinic.Methods Body, thymus and spleen weight of Npc1-/ -and Npc1+/ + mice aged (14±2) days, (42±2) days and (63±2) days (Day14±2 , Day42±2 and Day63±2 ) were recorded and the associated organ index were calcu-lated. White blood cell count in peripheral blood of mice aged Day42±2 was examined by routine blood test. Expression of cytokines at mRNA level in mouse peripheral blood was detected by qPCR. Percentages of CD4+, CD8+ and CD19+ lymphocytes in peripheral blood and spleen of mice aged Day42±2 were measured by flow cytometry. Apoptosis and senescence of spleen in mice aged Day63±2 were examined by immunofluores-cence and β-galactosidase staining. Results Compared with Npc1+/ + mice, there was no significant differ-ence in the weight of spleen and thymus in Npc1-/ - mice aged Day14±2; the weight of spleen in Npc1-/ - mice aged Day42±2 significantly increased, but the weight of thymus showed a significant decrease; furthermore, both the weight of spleen and thymus in Npc1-/ - mice aged Day63±2 significantly decreased; and the body weight of Npc1-/ - mice of each age group significantly decreased. Moreover, compared with Npc1+/ + mice, the absolute number of lymphocytes in the peripheral blood of Npc1-/ - mice aged Day42±2 showed no signifi-cant difference, but the percentage in whole white blood cells significantly decreased due to the significantly increased neutrophils. Expression of cytokines ( IL-1, IL-2, IFN-γ, TNF-α, IL-4, granzyme A and granzyme B) at mRNA level in the peripheral blood leukocytes of Npc1-/ - mice aged Day42±2 was abnormal as compared with that in Npc1+/ + mice. The number of T (CD4+ and CD8+) lymphocytes in Npc1-/ - mice aged Day42±2 significantly decreased, while the number of B (CD19+) lymphocytes increased significantly as com-pared with those in the Npc1+/ + mice. Compared with Npc1+/ + mice, apoptosis and senescence of the spleen in Npc1-/ - mice aged Day63±2 aggravated significantly. Conclusion The abnormal lipid metabolism triggered by Npc1 gene mutation causes severe immune dysfunction in Npc1-/ - mice. Therefore, immune dysfunction should be taken into full consideration when treating patients with NPC1, which might help improve the life quality and prolong the survival time.

Leptin, an adipocyte-derived peptide hormone, has been shown to facilitate breathing. However, the central sites and circuit mechanisms underlying the respiratory effects of leptin remain incompletely understood. The present study aimed to address whether neurons expressing leptin receptor b (LepRb) in the nucleus tractus solitarii (NTS) contribute to respiratory control. Both chemogenetic and optogenetic stimulation of LepRb-expressing NTS (NTS^LepRb) neurons notably activated breathing. Moreover, stimulation of NTS^LepRb neurons projecting to the lateral parabrachial nucleus (LPBN) not only remarkably increased basal ventilation to a level similar to that of the stimulation of all NTS^LepRb neurons, but also activated LPBN neurons projecting to the preBötzinger complex (preBötC). By contrast, ablation of NTS^LepRb neurons projecting to the LPBN notably eliminated the enhanced respiratory effect induced by NTS^LepRb neuron stimulation. In brainstem slices, bath application of leptin rapidly depolarized the membrane potential, increased the spontaneous firing rate, and accelerated the Ca²⁺ transients in most NTS^LepRb neurons. Therefore, leptin potentiates breathing in the NTS most likely via an NTS-LPBN-preBötC circuit.
Leptin/pharmacology* ; Membrane Potentials ; Neurons/metabolism* ; Solitary Nucleus/metabolism*