Objective To clarify the cellular localization of triptolide and to explore its in-cell action sites.Methods 4-(Bro-momethyl)-7-methoxycoumarin was employed to label triptolide,then labelled triptolide was incubated with human hepatoma carci-noma cells.Subsequently,incubated cells were subjected to stain with fluorescent dye DiI or PI,which were specific to cytoplasmic membrane system and nucleus,respectively.Results Compared with the non-triptolide control,coumarin labelled triptolide shown a light blue fluorescence under UV excitation;Co-localization with DiI showed that triptolide exist in cytoplasm and(or)on cell mem-brane;Co-localization with PI showed that triptolide located in cell nucleus.Moreover,microscopic observation indicated that the fluorescence intensity in nucleus was denser than that in cytoplasm.Conclusion The presnt study demonstrate that triptolide main-ly act in nucleus,followed by acting in cytoplasm and(or)on cell membrane.

A 28-year-old female patient presented with indurated erythema and nodules on the right lower limb for 2 years,with mild itching and pain.Skin examination showed a well-circumscribed irregular dark red patch measuring about 10 cm × 5 cm in size on the extensor aspect of the right thigh.On the patch,there were scattered or densely distributed mung bean-to soybean-sized quasi-circular violaceous nodules with a smooth surface,which were hard on palpation.Subcutaneous nodules with medium hardness were found on palpation,and hyperpigmentation was observed on the surface of some nodules.Local skin temperature was increased,with tenderness on palpation.Histopathologically,mononuclear cells showed nodular or sheet-like distribution in the middle and upper dermis,some of which had pale-staining cytoplasm.Moreover,plenty of plasma cells were observed.Immunohistochemistry revealed that histiocytes were stained strongly positive for S100.The number of IgG4-positive plasma cells increased obviously,and was more than 50 per high-power field (× 200).The proportion of IgG4-positive plasma cells in IgG-positive plasma cells was 45％.Finally,the patient was diagnosed with cutaneous Rosai-Dorfman disease with increased IgG4-positive plasma cells.

BACKGROUND: It has been believed mesenchymal stem cells (MSCs) play a role in treatment through paracrine mechanism. Various side effects such as embolism, tumorigenesis and immunological reaction caused by direct injection of MSCs can be avoided by extracting MSC lysate. However, there is a larger difference in current collection methods and standards of MSC lysate. OBJECTIVE: To compare repeated freeze-thaw and ultrasonication for the collection of lysate of MSCs. METHODS: Adipose-derived mesenchymal stem cells (ADMSCs) were isolated from the abdominal subcutaneous fat of healthy individuals, and purified with adherence screening method, followed by in vitro amplification using fetal bovine serum medium. The common surface makers of these cells were tested by flow cytometry (1×109, 2×109, 4×109/L). Repeated freeze-thaw and ultrasonication were employed for cell cytoclasis at three different densities respectively in saline and double distilled water, and a comprehensive comparison was performed on cytoclasis rate and the content of protein in cell lysate between the two methods. RESULTS AND CONCLUSION: (1) ADMSCs obtained from in vitro isolated human adipose tissue grew in a swirl or radial pattern with a homogenous size and neat arrangement. CD44, CD90, CD105 and other commonly used surface markers were highly expressed. (2) The study for optimization of lysate collection revealed that the higher cell density implicated a longer time for cell wall disruption and cytoclasis, as well as significantly increased cytoclasis rate. (3) BCA protein assay showed that the highest content of protein was obtained in saline solvent using ultrasonication method. Comprehensive analysis on the results leads to a conclusion that ultrasonication method with saline as the solvent is the optimized method for extraction of ADMSCs lysate, and the cell concentration of less than 4×109/L is recommended.

Objective To explore the characteristics of respiratory parameters in patients with different body mass index during general anesthesia with tracheal intubation. Methods 102 patients scheduled for otitis me-dia surgery were divided into low weight group(B1,n=32),normal weight group(B2,n=36)and overweight or obese group(B3,n = 34 ). After general anesthesia with tracheal intubation,the tidal volume of anesthetic ma-chine wasadjusted to maintain the end tidal carbon dioxide partial pressure between 35 - 45 mmHg. At 10 min (T1),30min(T2)and 60 min(T3)after adjustment,arterial PH,arterial partial pressure of oxygen(PaO2),arte-rial carbon dioxide pressure(PaCO2),inspiratory tidal volume(VTi),expiratory tidal volume(VTe),end tidal carbon dioxide partial pressure(PETCO2),peak airway pressure(Ppeak),plateau airway pressure(Pplat)and dy-namic lung compliance(Cdyn)were recorded. Results PH and PaO2 were not significantly different at T1-3 among the three groups(P>0.05). As compared with group B1 and B2,PaCO2 was lower in group B3. In comparison with group B2,VTi,VTe and Cdyn were higher in group B1 and lower in group B3(P < 0.05). Ppeak and Pplat were lower in group B1 but higher in group B3(P<0.05). PETCO2 was higher in group B1(P>0.05)while lower in group B3 (P < 0.05). Conclusions With the increase in BMI during general anesthesia with tracheal intubation ,the VTi,VTe,Cdyn,PETCO2 and PaCO2 decrease significantly,but Ppeak and Pplat elevate markedly. BMI is a refer-ence index for setting respiratory parameters.

Objective:To analyze the clinical value of ultrasonic examination in the diagnosis of alcoholic liver disease.Methods:From April 2015 to December 2017, 68 patients with suspected alcoholic liver disease in the Mental Health Center of Yiwu were examined by ultrasound and liver function test.The diagnostic value of ultrasound examination was analyzed.Results:Liver function examination showed that 36 cases were diagnosed as alcoholic liver disease, 32 cases were diagnosed as fatty liver, 12 cases were misdiagnosed and 12 cases missed diagnosis.Ultrasound examination showed that 47 patients were diagnosed as alcoholic liver disease, 21 patients were diagnosed as fatty liver, 1 patient was misdiagnosed and 2 patients were missed diagnosis.The sensitivity, specificity and total accuracy of routine liver function examination in diagnosis of alcoholic liver disease were 62.50%(30/48), 70.00%(14/20), 64.71%(44/68), respectively.The sensitivity, specificity and total accuracy of ultrasound in diagnosis of alcoholic liver disease were 95.83%(46/48), 95.00%(19/20), 95.59%(65/68), respectively.The sensitivity, specificity and total accuracy of ultrasonic examination in diagnosis of alcoholic liver disease were significantly higher than those of routine liver function examination (χ 2=16.168, 4.329, 20.379, all P<0.05). Ultrasound examination showed that 35 cases were alcoholic fatty liver, 8 cases were alcoholic hepatitis and 4 cases were alcoholic cirrhosis.The coincidence rate of diagnosis was 97.92%(47/48). The Kappa test value was 0.895, which indicated that the consistency was good. Conclusion:Ultrasonic examination is effective and accurate in diagnosis of alcoholic liver disease, and it can be used to diagnose the type of disease.

OBJECTIVETo observe the effect of ultrasound stimulation at the acupoint Guanyuan (CV 4) on follicular development in menopausal rats.

METHODSMenopausal female SD rats were selected by vaginal smear examinations. The rats were subjected to ultrasound stimulation at the acupoint Guanyuan with the output power of 0.1 W, working frequency of 9 MHz, and focal length of 4.5-5 mm. Electrochemiluminescence immunoassay was used to detect the serum estrogen levels of the menopausal rats. The changes in the ovarian tissue histology and the follicle number were observed.

RESULTSCompared with the control group, a 10-day ultrasound stimulation for 10 and 5 min daily at Guanyuan significantly increased the serum estrogen levels and the numbers of primary and secondary follicles (P<0.05) and reduced the number of atretic follicles in the menopausal rats (P<0.05).

CONCLUSIONUltrasound stimulation at the acupoint Guanyuan can increase the estrogen secretion function and promote the development of follicles in menopausal rats.

Acupuncture Points ; Animals ; Estrogens ; blood ; Female ; Menopause ; Ovarian Follicle ; growth & development ; Rats ; Rats, Sprague-Dawley ; Sonication

Objective To investigate the expression of DKK3 in human cutaneous malignant melanoma cells and tissues,and to evaluate the effect of transfection with DKK3 gene on migration and invasion of a malignant melanoma cell line A375.Methods Western blot analysis was performed to measure the relative expression of DKK3 in human cutaneous melanoma cell lines HM,A375,WM451,SK-MEL-1,Hs-695T,MDA-MB-435s and WM35,as well as pigmented nevus tissues.Real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of DKK3 in 58 melanoma tissues (including primary melanoma and metastatic melanoma) and 30 pigmented nevus tissues from Chongqing Traditional Chinese Medicine Hospital between August 2014 and June 2017.The pcDNA3.1 (+)-Flag-Vector (control group) and pcDNA3.1 (+)-Flag-DKK3 (transfection group) were transfected into A375 melanoma cells separately.RT-PCR and Western blot analysis were performed to verify the overexpression of DKK3,and to evaluate the effect of DKK3 overexpression on the expression of molecules related to the migration and invasion of melanoma cells.Cell scratch assay,Transwell migration and invasion assay were conducted to assess the effect of DKK3 on the migration and invasion of A375 cells.Statistical analysis was done by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and least significant difference (LSD)-t test for multiple comparisons with the SPSS 13.0 software.Results DKK3 protein was absent or lowly expressed in the human melanoma cell lines,but highly expressed in the pigmented nevus tissues.There were significant differences in the mRNA expression of DKK3 among the primary melanoma tissues (2-ΔΔCt:[0.325 ± 0.150] × 10-3),metastatic melanoma tissues ([0.142 ± 0.210] × 103) and pigmented nevus tissues ([0.634 ±:0.120] × 10-3,F =46.57,P ＜ 0.05).In addition,the mRNA expression of DKK3 was significantly lower in the metastatic melanoma tissues than in the primary melanoma tissues and pigmented nevus tissues (LSD-t =2.48,3.12,both P ＜ 0.05).After transfection with DKK3,cell scratch assay showed that the migration rate was significantly lower in the transfection group (22.11％ ± 5.11％) than in the control group (54.36％ ± 23.22％,t =2.36,P ＜ 0.001).Transwell migration and invasion assay revealed that the number of A375 cells crossing the Transwell chamber was significantly lower in the transfection group (265 ± 33,76 ± 18 respectively) than in the control group (429 ± 41,135 ± 21 respectively;t =1.24,1.35 respectively,both P ＜ 0.001).After overexpression of DKK3 in the A375 cells in the transfection group,the mRNA and protein expression of E-cadherin were up-regulated,while the mRNA and protein expression of N-cadherin,vimentin,matrix metalloproteinase 2 (MMP2),MMP7 and MMP11 were down-regulated compared with the control group.Conclusions The expression of DKK3 is down-regulated in the melanoma cell lines and tissues,and the migration and invasion of A375 cells are markedly inhibited by overexpression of DKK3.DKK3 may be a target for inhibiting the metastasis of cutaneous malignant melanoma.

OBJECTIVE：To interpret the new version of the Implementation Plan for Enrollment and Assessment of Clinical Pharmacist Teacher Training Project of Chinese Hospital Association （Trial）（hereinafter referred to as the “New Plan ”）officially announced by the Chinese Hospital Association in Sept. 2021，in order to provide guidance and reference for smoothly promoting the implementation of the New Plan and realizing the original intention of the reform. METHODS ：The New Plan was interpreted from four aspects ，including the background of the formulation of the New Plan ，the main features presented ，the innovations and the issues that need to be paid attention to in the next implementation. RESULTS & CONCLUSIONS ：The formulation and release of the New Plan marks the beginning of a new round of reform of the clinical pharmacist teacher training program of the Chinese Hospital Association. It is mainly based on the actual problems encountered in the operation of the current clinical pharmacist teacher training system ，the importance of clinical pharmacistteacher training itself ，and the opportunities and challenges that the development of the times has brought to our country ’s 163.com clinical pharmacy business. Research foundation ， problem orientation and professional demonstration present important characteristics. Compared with the old plan ，the New Plan adds a substantive selection mechanism for majors ，which intends to achieve the necessary “strictness”in the enrollment process ；it reconstructs the overall training goal of the benchmarking project and the three-in-one assessment system ，which intends to achieve a reasonable “lax”of assessment link ；it removes restrictions on off-site training ，advocates wide-ranging professional docking，and improves the flexibility of the training system and process management. The implementation of the New Plan is a complex work involving multiple parties and is affected by many factors. It requires multiple teams to perform their duties and promote together. The teacher training base and expert team must understand the spirit of reform and achieve the stable implementation of the New Plan ；at the same time ，the communication between the project working group and the teaching base and experts should be strengthened ，and finally realize the original intention of the reform of cultivating more high-quality clinical pharmacy teachers.