Objective To establish a model for studying CD8+ cytotoxic T lymphocyte proliferation in vitro and to screen traditional Chinese drugs (TCDs) with immunosuppressive effects.Methods Spleen tissue was isolated from mice,and made into single cell suspensions followed by separation of CD8+ T lymphocytes with specific antibodies.Then,the CD8 + T lymphocytes were seeded into anti-CD3/CD28 antibody-coated 96-well plates and cocultured with the extracts of 23 TCDs (100 mg/L) separately for 96 hours.Those ceils cultured with and without the presence of anti-CD3/CD28 antibody alone served as the positive control and negative control respectively.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to evaluate the proliferation of cells and to select the top four TCDs with the strongest inhibitory effect.The relationship between the inhibitory effect and TCD concentrations was further assessed for the four selected TCDs.Enzyme-linked immunospot (ELISPOT) assay was carried out to estimate the influence of the four TCDs on the secretion of interferon (IFN)-γ by CD8+ T lymphocytes induced by anti-CD3/CD28 antibodies.Statistical analysis was done by nonparametric rank sum test.Results Of the 23 TCDs,14 significantly inhibited the proliferation of CD8+ T lymphocytes (all P ＜ 0.05),of which,Rhizoma Coptidis,Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae displayed the strongest inhibitory capacity with the 50％ inhibitory concentration being 25,35,50 and 60 mg/L respectively,and the 100％ inhibitory concentration being 200,100,200 and 200 mg/L respectively.The anti-CD3/CD28 antibody-induced secretion of IFN-γby CD8+ T lymphocytes was markedly suppressed by Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae at the concentration of 100 mg/L,but not by Rhizoma Coptidis at this concentration.Conclusions A model for studying the proliferation of CD8+ T lymphocytes is successfully developed in vitro,and four TCDs with strong inhibitory effects on the proliferation of CD8+ T lymphocytes have been screened out with this model.

Aim To study the effect of halometasone in combination with scutellaria baicalensis georgi on the vitiligo mice induced by monobenzone. Methods 40% monobenzone cream was applied to induce vitiligo in C57BL/6 mice. Through the halometasone, halo-metasone and scutellaria baicalensis georgi combined with 40% monobenzone cream, the influence of halo-metasone and scutellaria baicalensis georgi on mice de-colorizing was studied. Hair decolorizing was observed with the naked eye, the skin decolorizing was observed by reflectance confocal microscopy ( RCM ) , and CD8 +T cell infiltration was tested with immunofluores-cence detection. The serum levels of interleukin-6(IL-6 ) and tumor necrosis factor-α( TNF-α) were deter-mined by enzyme linked immunosorbent assay ( ELISA) . Results Mice in model group showed de-pigmentation at both the monobenzone application part and non-application part. The halometasone group did not show significant therapeutic efficacy. In halometa-sone and scutellaria baicalensis georgi treatment group, there was less decolorization, the occurrence ratio, the scores of occurring time and size were lower compared with model group. There were fewer infiltrated lympho-cytes and CD8 +T cells. Halometasone and scutellaria baicalensis georgi group also showed that the serum levels of IL-6,TNF-α decreased. Conclusion Halo-metasone and scutellaria baicalensis georgi have thera-peutic effect on vitiligo mice induced by monobenzone.

Objective To determine the clinical features of childhood vitiligo in Han nationality.Methods A standardized questionnaire was used to clinically investigate 396 cases of vitiligo in patients aged less than 12 years;825 adult patients with vitiligo served as controls.Data analysis was done by SPSS 10.0.Results In child patients with vitiligo,46.2% were males,and 53.8%females;there was no statistically significant difference in gender ratio between the childhood and adult groups(P＜0.05).The incidence of segmental vitiligo was 33.3%in children,significantly higher than that in adult patients(13.5%).On the contrary,the incidence of generalized vitiligo was significantly lower in children han in adult patients(4.3%vs 11.6%).A family history of vitiligo was observed in 12.4%of children,and in 12.7% of adults,withoutsignificant difference between the two groups.For both childhood and adult vitiligo patients,the incidence of vitiligo was significantly higher in firSt-degree relatives than in second-degree relatives.The most common precipitating factor of childhood vitiligo was trauma.The incidence of halo nevi was higher in patients with childhood vitiligo compared to patients with adult vitiligo,while thyroid disease was seen more frequently in adult patients.Conclusion There are some differences in clinical characteristics of vitiligo between children and adults.

Objective To analyse the possible risk factors associated with the progression of vitiligo.Methods A questionnaire survey was carried out to collect the clinical data on 1088 patients with vitiligo.The relationship between possible inducements to the progression of vitiligo and lesion area was statistically analyzed in patients with the same clinical course of vitiligo. Paired t test was performed to compare the mean area index of involvement between patients with inducements and those without Results A significant difference was observed in the mean area index of involvement between patients with isomorphic response and those without (t = 6.770, P ＜ 0.01 ) as well as between patients negatively affected by psychiatric factors and those unaffected (t = 6.704, P ＜ 0.01 ), but not between patients with family history and those without (t = 1.499,P ＞ 0.05). Conclusion A rapid progression of vitiligo is more likely to be observed in patients negatively affected by psychiatric factors and patients with isomorphic response.

Objective To study the clinical and electrophysiological features of the patients with hereditary neuropathy with liability to pressure palsy ( HNPP) diagnosed by gene analysis.Methods Seven patients from two HNPP families were assessed on medical history, physical examination, electrophysiology findings and gene analysis.Results A clinical manifestation of acute, painless, recurrent peripheral nerve palsies was typical for HNPP.Median, ulnar and peroneal nerves were usually affected.Electrophysiology study revealed that prolonged distal motor latency and slowing nerve conduction velocity were prominent.Gene studies exhibited a deletion of the peripheral myelination protein 22 gene in all the seven patients.Conclusions HNPP usually affects areas where nerves are subject to entrapment, and many episodes are preceded by minor compression on the affected nerve.As a reliable screening tool in detecting HNPP, the electrophysiological study shows that segmental demyelination is most commonly seen at common nerve entrapment sites.

Objective To investigate the mechanism of repigmentation in vitiligo induced by narrow band ultraviolet B (NB-UVB) via observing the effects of 311 nm NB-UVB on the proliferation of, apoptosis and melanogenesis in melanocytes. Methods Immortalized B10BR melanocytes were irradiated with NB-UVB at varying doses (400, 800 and 1200 mJ/cm2). Subsequently, the proliferation of and apoptosis in melanocytes were detected by MTT assay and flow cytometry, respectively, and the content of melanin was determined by NaOH assay. The expression of BCL-2 mRNA was examined by RT-PCR, and MC-1R expression of melanoeytes by immunocytochemistry and Western blotting. Results The proliferation of and apoptosis in melanocytes experienced no obvious change after irradiation with NB-UVB at the three tested doses. In B10BR melanocytes irradiated with NB-UVB at 400, 800 and 1200 mJ/cm2, the melanin content was 1.42, 1.78, 2.05 times, the mRNA expression of BCL-2 was 1.75, 2.32, 3.28 times, and the protein expression of MC-1R was 1.68, 2.35 and 3.01 times, that in unirradiated melanocytes, respectively. Conclusion NB-UVB irradiation at therapeutic doses could promote the melanogenesis in, enhance anti-oxidative stress activities of melanocytes,by upregulating the expressions of BCL-2 and MC-1R, with no marked effects on the apoptosis in melanocytes.

Objective To develop a HPLC method for determination of baicalin .Methods The separation was carried out on a Waters XBridge C18 column(4 .6 mm × 250 mm ,5μm) ,the mobile phase was composed of acetonitrile and 0 .8% for-mic acid (25∶75) ,the detection wavelength was set at 276 nm ,the flow rate was 1 .0 ml/min ,the column temperature was 30 ℃ and the injection volume was 10 μl .Results The linearity was obtained over 1 .25-40 μg/ml(r=0 .999 9) for baicalin . The RSD of precision were less than 2% .The average recovery was between 95% and 100% .Conclusion This HPLC method was simple ,accuracy and suitable for the quality control of Biyanling capsule .

Objective To establish a convenient ~(13)C-breath test system in live mice,and investigate the value of ~(13)C-methacetin breath test(~(13)C-MBT) in the diagnosis of acute liver damage of mice with domestically synthesized ~(13)C-methacetin. Methods Domestically synthesized ~(13)C.methacetin was prepared from aeamol by methylation. Abdominal injection of CCl_4 was adopted to duplicate acute liver damage of mice,then the mice were housed under normal laboratory condition for a whole month to gain recovery,which were indentified by hepatic pathological examinations and biochemical teats of liver function.After fasting, the mice were orally administered ~(13)C-methacetin,and the expired air was collected at various time points. Infrared spectrometer was employed, and delm over baseline(DOB) curves of ~(13)C-exhalation were drawn. Results Six to eight min after administration of ~(13)C-methacetin,the rate of ~(13)C-exhalation peaked in control group(51.9±2.04), and decreased thereafter. Sixteen min after administration of ~(13)C-methacetin,the rate of ~(13)JC-exhalation peaked in model group(26.37±5.74), and decreased thereafter.There were significant differences between these two groups(P<0.05).There was no significant difference in peak value and time to reach the peak on DOB curves of ~(13)C-methacetin breath test after the two groups of mice were housed under the same condition for a month(P>0.05).Conclution ~(13)C-MBT facilitates the collection and evaluation of ~(13)CO_2 in the expired air of live mice,and yields precise reflection of alterations of liver function in acute liver injury and functional recovery.

OBJECTIVE: To study the relationship between fibrinogen level and pathogenesis of sudden sensorineural hearing loss(SSHI.). METHOD: Fifty patients (55 ears) with SSHL within 7 days of the onset were studied: a control group was consist of 50 normal-hearing people who were individually matched on a pairwise basis according to the same gender and age. Both the patients and the normal people were tested for the parameters of hemorheology, blood biochemistry, whole blood cell count and clotting function. RESULT: Fibrinogen level and plasma viscosity in patients with SSHL were significantly higher than that in control subjects. Prothrombin time and activated partial thromboplastic time were significantly less in the patients group than that in the control group (P < 0.05). There were statistical difference. The parameters of blood biochemistry, whole blood cell count and platelet adhesion test of two groups had no significant difference (P > 0.05). CONCLUSION Elevated plasma fibrinogen may be a major pathogenesis of SSHL. An increase in plasma fibrinogen level may lead to elevated plasma viscosity. All these may promote a prothrombin or hypercoagulable state and impair blood perfusion of cochlea.
Adolescent ; Adult ; Aged ; Case-Control Studies ; Female ; Fibrinogen ; metabolism ; Hearing Loss, Sensorineural ; blood ; etiology ; Hearing Loss, Sudden ; blood ; etiology ; Hemorheology ; Humans ; Male ; Middle Aged ; Young Adult

Objective : To investigate the effect of transforming growth factor β1 (TGF-β1) on human periodontal ligament fibroblasts (hPDLFs) stimulated by lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS). Methods: hPDLFs were obtained and identified by immunohistochemistry. The stimulating concentration of Pg-LPS was determined by qRT-PCR and CCK-8. The hPDLFs were divided into 4 groups: blank control group, 100 μg/mL pure Pg-LPS; low concentration group, 1 ng/mL TGF-β1+100 μg/mL Pg-LPS; medium concentration group, 10 ng/mL TGF-β1+100 μg/mL Pg-LPS; and high concentration group 100 ng/mL TGF-β1+100 μg/mL Pg-LPS. Cell proliferation was measured by CCK-8 assay at 72 hours, cell migration was measured by scratch and Transwell chamber assays at 24 hours, and the cell cycle of the hPDLFs was measured by flow cytometry at 72 hours. The expression of Forkhead/winged helix transcription factor p3 (Foxp3), interleukin-6 (IL-6) and Epstein-Barr virus-induced gene 3 (EBI3) mRNA in hPDLFs at 72 hours was measured by qRT-PCR, and the expression of Foxp3, IL-6 and EBI3 proteins in hPDLFs at 72 hours was detected by western blot. Results : The immunohistochemistry results showed that anti-vimentin was positive and anti-keratin was negative. At a concentration of 100 μg/mL Pg-LPS, the expression of IL-6 mRNA in hPDLFs was increased (P<0.000 1), and the proliferation of hPDLFs was decreased (P<0.000 1). Therefore, 100 μg/mL PG-LPS was selected to simulate the inflammatory state. 10, 100 ng/mL TGF-β1 could improve the proliferation ability of hPDLFs in inflammatory state (P<0.000 1) ; 1, 10 and 100 ng/mL TGF-β1 could promote the migration ability of hPDLFs in inflammatory state (P<0.000 1). 1, 10 and 100 ng/mL TGF-β1 could accelerate the cell cycle of hPDLFs in inflammatory state (P<0.000 1). 1, 10 and 100 ng/mL TGF-β1 could inhibit the expression of IL-6 gene and protein in hPDLFs in inflammatory state (P<0.000 1), 1 and 10 ng/mL TGF-β1 could increase the expression of EBI3 gene and protein in hPDLFs in inflammatory state (P<0.000 1). 1, 10 ng/mL TGF-β1 could increase the expression level of Foxp3 gene in hPDLFs in inflammatory state, and 10 ng/mL TGF-β1 could increase the expression level of Foxp3 protein (P<0.05). Conclusion TGF-β1 can promote the proliferation and migration of hPDLFs under inflammatory conditions, upregulate EBI3 and inhibit inflammation, which may be related to the expression of the transcription factor Foxp3.