Objective To investigate the effect of calcium ions on the expression of kallikrein-4(KLK4)and cell growth of ameloblast,and to provide an experimental basis for calcium ion promoting normal mineralization of enamel.Methods ALC cells were treated with 0,2.0,2.5,3.0,and 3.5 mmol/L CaCl2 for 24 and 48 h.KLK4 expression was analyzed by qRT-PCR and Western blot analysis.The viability of ALC cells was determined by using CCK-8.Annex-inV-FITC/PI dual staining combined with flow cytometry and Hoechst 33342 staining were used to detect the ALC cell cycle and cell apoptosis.The protein expression level of glucose-regulated protein 78(GRP78)was measured by West-ern blot analysis.Results After 24 h of treatment with 2.5,3.0,and 3.5 mmol/L CaCl2,the expression of KLK4 mRNA was increased(P＜0.05),and after 24 h of treatment with 2.0,2.5,3.0,and 3.5 mmol/L CaCl2,the expression of KLK4 protein was increased(P＜0.05).After 48 h of treatment with 3.0 mmol/L and 3.5 mmol/L CaCl2,the expression of KLK4 mRNA and protein was increased(P＜0.05).Compared with the control group,the viability of ALC cells was in-creased after 24 and 48 h of treatment with 2.0,2.5,and 3.0 mmol/L CaCl2(P＜0.05),and the highest cell viability was observed with 2.5 mmol/L CaCl2.Hoechst 33342 staining results showed that 3.0 mmol/L and 3.5 mmol/L CaCl2 may promote apoptosis in ALC cells.Flow cytometry showed that the proportion of G2/M phase cells and the apoptosis rate increased after 3.5 mmol/L CaCl2 treatment for 24 h(P＜0.05),compared with the 0,2.0,2.5,and 3.0 mmol/L CaCl2 groups.After 24 h of treatment with 3.0 mmol/L and 3.5 mmol/L CaCl2,the expression of GRP78 protein was re-duced(P＜0.05),and after 48 h of treatment with 2.5 mmol/L CaCl2,the expression of GRP78 protein was reduced(P＜0.05).Conclusion Calcium ions can promote the increase of KLK4 expression and cell viability in ALC cells,but a higher concentration of calcium ions can block the G2/M phase of ALC cells,thus inducing apoptosis of ALC cells and reducing the expression of apoptosation-related protein GRP78.

[Objective] To investigate the effects of fluoride on kallikrein-4 (KLK4) and cell apoptosis as well as the possible mechanisms in ALC cells. [Methods] ALC cells were treated with different concentrations of fluoride for 24 and 48 hours. The effects on cell viability, cell cycle and cell apoptosis were detected using CCK-8, flow cytometry and hoechst 33342, respectively. KLK4 expression was detected by qRT-PCR and Western blotting, and the protein expressions of glucose-regulated protein 78 (GRP78), p-eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were detected by Western blotting. [Results] The results showed that the expression of KLK4 was significantly reduced after treatment with 1.0 and 2.0 mmol/L NaF for 24 and 48 hours (P<0.05). The difference was statistically significant in cell activity between 2.0 mmol/L NaF treatment group and the control group (P<0.05). The G0/G1 phase cells significantly reduced and the S phase cells significantly increased after treatment with 0.1 and 1.0 mmol/L NaF for 24 hours (P<0.05), while the G0/G1 phase cells significantly increased and the S phase cells significantly reduced in the 2.0 mmol/L NaF treatment cells (P<0.05). The G0/G1 phase cells significantly increased and G2/M phase cells significantly reduced after treatment with 2.0 mmol/L NaF for 48 hours (P<0.05). With the increase of NaF treatment concentration, the number of bright blue cells gradually increased, and the percentage of apoptosis also increased successively. Except for the cells treated with 0.1 mmol/L NaF for 24 hours, the apoptosis rate of the other fluoride treatment groups was statistically significant compared with the control group (P<0.05). The expressions of GRP78 and p-eIF2α were significantly increased after treatment with 0.1, 1.0, and 2.0 mmol/L NaF for 24 hours (P<0.05). The expressions of ATF4 and CHOP were significantly increased after treatment with 1.0 and 2.0 mmol/L NaF for 24 hours (P<0.05). The expressions of ATF4 and CHOP were significantly increased after treatment with 0.1, 1.0, and 2.0 mmol/L NaF for 48 hours (P<0.05). [Conclusion] Sodium fluoride may result in inhibition of KLK4 expression and abnormal cell growth, possibly by inducing GRP78 expression and activating eIF2α/ATF4/CHOP signaling pathway in ALC ameloblasts.

Objective: To observe the effect of calcium on biological characteristics (proliferation, apoptosis and cell cycle) of ALC ameloblasts. . Methods: ALC cell lines were cultured in vitro in DMEM medium with high glucose at different concentrations (0, 2.0, 2.5, 3.0 and 3.5 mmol/L CaCl2 aqueous solution) for 24 h and 48 h, respectively. Changes of ALC cells under two kinds of incubation time were observed with an inverted microscope. CCK-8 method was used to analyze the effect of calcium ion on ALC cell proliferation. Hoechst staining was used to observe the effect of calcium ion on ALC cell apoptosis. PI staining and FCM method were used to analyze the effect of calcium ions on the growth cycle of ALC cells. Western blot was used to detect the effect of calcium ions on the expression of Cyclin A, Cyclin B and Cyclin D in ALC cells Results: In the 0 mmol/L CaCl2 group, ALC cells were oval or polygonal in shape, and the cells were closely connected and grew like paving stones. In other concentration groups, the morphology of ALC cells did not change significantly after calcium intervention for 24 h and 48 h. Results of CCK-8 method showed that the survival rate of ALC cells slightly decreased with increasing calcium ions concentration after calcium intervention for 24 h and 48 h. However, there was no significant differences in this trend. Results of Hoechst staining showed that the number of ALC cell apoptosis did not increase significantly after different concentrations of calcium intervention for 24 h and 48 h. With the increase of calcium ion concentration, results of PI staining and FCM method showed that the cell cycle of ALC cells gradually increased in S phase and decreased in G1 and G2 phase gradually. Western blot results showed that the expression of Cyclin A and Cyclin B in ALC cells decreased and the expression of Cyclin D increased after different concentrations of calcium intervention for 24 h and 48 h. Conclusion In this study, calcium has no significant effect on the proliferation and apoptosis of ALC cells. Calcium, however, has an effect on the ALC cell cycle. Results of this study show that calcium ions has no obvious toxic or side effects on the ameloblasts, which could be used to explore the possible mechanism and effect of calcium on dental fluorosis.

Objective:To compare the differences in the prevalence of mild micro-hepatic encephalopathy (MHE) among patients with cirrhosis by using the psychometric hepatic encephalopathy score (PHES) and the Stroop smartphone application (Encephal App) test.Methods:This prospective, multi-center, real-world study was initiated by the National Clinical Medical Research Center for Infectious Diseases and the Portal Hypertension Alliance and registered with International ClinicalTrials.gov (NCT05140837). 354 cases of cirrhosis were enrolled in 19 hospitals across the country. PHES (including digital connection tests A and B, digital symbol tests, trajectory drawing tests, and serial management tests) and the Stroop test were conducted in all of them. PHES was differentiated using standard diagnostic criteria established by the two studies in China and South Korea. The Stroop test was evaluated based on the criteria of the research and development team. The impact of different diagnostic standards or methods on the incidence of MHE in patients with cirrhosis was analyzed. Data between groups were differentiated using the t-test, Mann-Whitney U test, and χ2 test. A kappa test was used to compare the consistency between groups. Results:After PHES, the prevalence of MHE among 354 cases of cirrhosis was 78.53% and 15.25%, respectively, based on Chinese research standards and Korean research normal value standards. However, the prevalence of MHE was 56.78% based on the Stroop test, and the differences in pairwise comparisons among the three groups were statistically significant (kappa = -0.064, P < 0.001). Stratified analysis revealed that the MHE prevalence in three groups of patients with Child-Pugh classes A, B, and C was 74.14%, 83.33%, and 88.24%, respectively, according to the normal value standards of Chinese researchers, while the MHE prevalence rates in three groups of patients with Child-Pugh classes A, B, and C were 8.29%, 23.53%, and 38.24%, respectively, according to the normal value standards of Korean researchers. Furthermore, the prevalence rates of MHE in the three groups of patients with Child-Pugh grades A, B, and C were 52.68%, 58.82%, and 73.53%, respectively, according to the Stroop test standard. However, among the results of each diagnostic standard, the prevalence of MHE showed an increasing trend with an increasing Child-Pugh grade. Further comparison demonstrated that the scores obtained by the number connection test A and the number symbol test were consistent according to the normal value standards of the two studies in China and South Korea ( Z = -0.982, -1.702; P = 0.326, 0.089), while the other three sub-tests had significant differences ( P < 0.001). Conclusion:The prevalence rate of MHE in the cirrhotic population is high, but the prevalence of MHE obtained by using different diagnostic criteria or methods varies greatly. Therefore, in line with the current changes in demographics and disease spectrum, it is necessary to enroll a larger sample size of a healthy population as a control. Moreover, the establishment of more reliable diagnostic scoring criteria will serve as a basis for obtaining accurate MHE incidence and formulating diagnosis and treatment strategies in cirrhotic populations.

Objective:To analyze the clinical effect of navigation-assisted cosmetic incision for reduction and internal fixation in treating unilateral B-type zygomatic fracture.Methods:A retrospective cohort study was performed on clinical data of 35 patients with unilateral type B zygomatic fracture treated from January 2018 to December 2019 in First Affiliated Hospital of Fujian Medical University. There were 20 males and 15 females at age range of 5-62 years [(38.7±11.3)years]. Navigation-assisted cosmetic incision for reduction and internal fixation was performed for 17 patients (navigation group), and empirical incision to reduction and internal fixation was performed for 18 patients (convention group). The length of bony zygomatic process (zygomatic process) and width of zygomatic temporal point (frontal width) of the bilateral zygomatic bone were measured on the horizontal axis of CT at 1 week after operation. The absolute values of the difference of bony zygomatic process degree and frontal bony width between affected side and the healthy side were compared between the two groups. The patients′ satisfaction and occurrence of complications such as lower eyelid ectropion, incision infection and facial nerve injury were compared between the two groups at half a year after operation.Results:All patients were followed up for 6-24 months [(9.3±1.2)months]. The absolute difference of bony zygomatic process was 0.60(0.25, 0.85) mm in navigation group, and was 0.75 (0.20, 1.98)mm in convention group ( P>0.05). The absolute difference of frontal bony width was (0.37±0.11)mm in navigation group, and was (2.47±0.63)mm in convention group ( P<0.01). Satisfaction rates by both objective evaluation and subjective evaluation in navigation group were better than that in convention group at half a year after operation (both P<0.05). Navigation group showed lower eyelid ectropion in 1 patient and incision infection in 1 patient. Convention group showed facial nerve injury in 1 patient and incision infection in 2 patients. There was no significant difference in the incidence of complications between navigation group [12%(2/17)] and conventional group [17%(3/18)] ( P>0.05). Conclusion:For unilateral type B zygomatic fracture, navigation-assisted cosmetic incision for reduction and internal fixation can more accurately restore the frontal width, and improve satisfaction rate as compared with empirical reduction and internal fixation.

Objective:To investigate the current status of prevention and treatment of esophagogastric variceal bleeding (EVB) in cirrhotic portal hypertension patients in Ningxia region.Methods:The retrospective and descriptive study was conducted. The clinical data of 820 cirrhotic portal hypertension patients who were admitted to 21 medical centers in Niangxia region from January 2018 to December 2020 were collected, including 85 cases in Ningxia Hui Autonomous Region People′s Hospital, 73 cases in the Fifth People′s Hospital of Ningxia Hui Autonomous Region, 59 cases in the Wuzhong People′s Hospital, 52 cases in the Qingtongxia People′s Hospital, 50 cases in the Guyuan People′s Hospital, 47 cases in the Yuanzhou District People′s Hospital of Guyuan City, 47 cases in the Yinchuan Second People′s Hospital, 40 cases in the General Hospital of Ningxia Medical University, 40 cases in the Tongxin People′s Hospital, 35 cases in the Yinchuan First People′s Hospital, 34 cases in the Third People′s Hospital of Ningxia Hui Autonomous Region, 32 cases in the Zhongwei People′s Hospital, 30 cases in the Lingwu People′s Hospital, 30 cases in the Wuzhong New District Hospital, 30 cases in the Yanchi People′s Hospital, 29 cases in the Ningxia Hui Autonomous Region Academy of Traditional Chinese Medicine, 28 cases in the Shizuishan Second People′s Hospital, 25 cases in the Shizuishan First People′s Hospital, 21 cases in the Haiyuan People′s Hospital, 20 cases in the Pengyang People′s Hospital, 13 cases in the Longde People′s Hospital. There were 538 males and 282 females, aged (56±13)years. Observation indicators: (1) clinical charac-teristics of cirrhotic portal hypertension patients; (2) overall prevention and treatment of EVB in cirrhotic portal hypertension patients; (3) prevention and treatment of EVB in cirrhotic portal hypertension patients from different grade hospitals. Measurement data with normal distribution were represented as Mean± SD. Count data were described as absolute numbers, and comparison between groups was analyzed using the chi-square test. Results:(1) Clinical characteristics of cirrhotic portal hypertension patients: of 820 cirrhotic portal hypertension patients, 271 cases were in compensated stage and 549 cases were in decompensated stage. Of the 271 cases in compensated stage, there were 183 maels and 88 females, aged (53±12)years. There were 185 Han people, 85 Hui people and 1 case of other ethic group. The etiological data of liver cirrhosis showed 211 cases of viral hepatitis B, 4 cases of alcoholic liver disease, 8 cases of viral hepatitis C, and 48 cases of other etiology. There were 235 cases of Child-Pugh grade A and 36 cases lack of data. Of the 549 cases in decompensated stage, there were 355 males and 194 females, aged (57±14) years. There were 373 Han people, 174 Hui people and 2 cases of other ethic group. The etiological data of liver cirrhosis showed 392 cases of viral hepatitis B, 33 cases of alcoholic liver disease, 10 cases of viral hepatitis C, and 114 cases of other etiology. There were 80 cases of Child-Pugh grade A, 289 cases of grade B, 170 cases of grade C and 10 cases lack of data. (2) Overall prevention and treatment of EVB in cirrhotic portal hypertension patients: of 271 patients in compensated stage, 38 cases received non-selective β-blocker (NSBB) therapy, 16 cases received endoscopic treatment, 6 cases received interventional therapy. Of 549 patients in decompensated stage, 68 cases received NSBB therapy, 46 cases received endoscopic treatment, 28 cases received interventional therapy. (3) Prevention and treatment of EVB in cirrhotic portal hypertension patients from different grade hospitals: of 271 patients in compensated stage, 181 cases came from tertiary hospitals, of which 28 cases received NSBB therapy, 15 cases received endoscopic treatment, 6 cases received interventional therapy. Ninety cases came from secondary hospitals, of which 10 cases received NSBB therapy, 1 cases received endoscopic treatment. There was no significant difference in NSBB for prevention of EVB between tertiary and secondary hospitals ( χ2=0.947, P>0.05), while there was a significant difference in endoscopic treatment for prevention of EVB between tertiary and secondary hospitals ( χ2=5.572, P<0.05). Of 549 patients in decompensated stage, 309 cases came from tertiary hospitals, of which 22 cases received NSBB therapy, 29 cases received endoscopic treatment, 22 cases received interventional therapy. Two hundreds and fourty cases came from secondary hospitals, of which 46 cases received NSBB therapy, 17 cases received endoscopic treatment, 6 cases received interven-tional therapy. There were significant differences in NSBB and interventional therapy for prevention of EVB between tertiary and secondary hospitals ( χ2=18.065, 5.956, P<0.05). Conclusions:The proportion of receiving EUB prevention in cirrhotic portal hypertension in Ningxia is relatively low. For patients with compensated liver cirrhosis, the proportion of NSBB therapy and endoscopic treatment in the secondary hospitals was lower than that in tertiary hospitals. For patients with decompensated liver cirrhosis, the proportion of interventional treatment in secondary hospitals is lower than that of tertiary hospitals, but the proportion of NSBB in secondary hospitals taking is higher than that of tertiary hospitals.

【Objective】 To explore the effect of low levels of calcium on the biological characteristics of ameloblasts. 【Methods】 Rat primary ameloblasts were cultured in standard DMEM medium. After five days they were identified by RT-PCR and immunohistochemistry. Then 0, 0.6 and 1.2 mmoL/L Ca²⁺ and 100 mL/L fetal bovine serum were added into DMEM medium without calcium. After 48 hours, the cell morphology was observed by inverted microscope. The proliferation and apoptosis of cells were separately examined by MTT and AnnexinV-PI. Real-time quantitative PCR was used to detect the expression levels of amelogenin and KLK4 mRNA. 【Results】 After Five days in standard DMEM medium, the cells were shaped like the paving pattern. RT-PCR showed that both amelogenin and KLK4 were expressed in the cells. Immunohistochemical staining showed that most cells had positive staining for amelogenin. After 48 hours of calcium intervention, some cells in 1.2 mmoL/L Ca²⁺ group had higher nuclear density and poor light transmittance, and more high columnar cells could be observed in 1.2 mmoL/L Ca²⁺ group than those in 0 and 0.6 mmoL/L Ca²⁺ groups. With the decrease in calcium concentration in the medium, MTT showed that the proliferation activity of ameloblasts reduced (P<0.01). Annexin V-PI showed that the percentage of apoptotic cells decreased, and there was a significant difference between 1.2 mmoL/L and 0 mmoL/L Ca²⁺ groups (P<0.05). Real time-PCR showed that the expressions of amelogenin and KLK4 mRNA reduced (P<0.01). 【Conclusion】 Low-level calcium may inhibit the differentiation of ameloblasts, thereby affecting the formation of enamel mineralization.

Objective:To determine whether amoxicillin had an effect on the enamel mineralization of SD rats.Methods:Eighteen SD rats were randomly divided into three groups. The rats in the control group were given distilled water. The rats in two experimental groups were administered 50 or 100 mg/kg amoxicillin by intragastric administration from day 3 to day 17 after birth. The general condition, the structure of liver and kidney, the enamel surface changes of mandibular first molars and incisors were observed. The changes of Ca/P ratio on enamel surface were analyzed by X-ray energy dispersive spectrometer (EDS). The surface morphology after phosphoric acid treatment was examined by scanning electron microscopy (SEM). Histological changes in the ameloblasts of mandibular incisors were analyzed by hematoxylin and eosin (HE) staining.Results:Compared with the control group, the general conditions as well as liver and kidney structures of SD rats in 50 and 100 mg amoxicillin groups had no significant differences. There was no obvious chalky changes on the first mandibular molars of SD rats in each group. All the incisors in 50 and 100 mg groups showed different degrees of chalkiness in the labial incisal 1/3 enamel. X-ray EDS analysis showed that the Ca/P ratios of occlusal and incisal 1/3 enamel in 50 and 100 mg groups (occlusal 1/3 of mandibular first molars: 1.51±0.03 and 1.52±0.02, incisal 1/3 of mandibular incisors: 1.46±0.01 and 1.43±0.01) was significantly lower than that in the control group (occlusal 1/3 of mandibular first molars: 1.67±0.41, incisal1/3 of mandibular incisors: 1.73±0.07) ( P<0.05). However, there was no significant differences in the cervical 1/3 Ca/P ratio of mandibular first molars and incisors among the three groups (mandibular first molars: 1.56±0.04 for control group, 1.59±0.05 for 50 mg group and 1.57±0.04 for 100 mg group; incisors: 1.52±0.02 for control group, 1.47±0.01 for 50 mg group and 1.51±0.03 for 100 mg group) ( P>0.05). SEM observation showed that the enamel rods of the first molars and incisors in the 50 and 100 mg group varied in size and arranged disorderly. The spaces between the enamel rods were larger than that in the control group and some areas even appeared large pits. HE staining showed that the gaps between ameloblasts in 50 and 100 mg groups were significantly wider than that in the control group. Conclusions:Intake of amoxicillin during the period of enamel development of SD rats might affect enamel mineralization.

Objective To investigate the suppressive effect of prostaglandin E2 (PGE2),hepatocyte growth factor (HGF) and indoleamine 2,3-dioxygenase (IDO) whose secretion was promoted by human umbilical cord mesenchymal stem cells(MSCs) on the activated peripheral blood CD4+ T cells in primary Sj(o)gren syndrome (pSS) in vitro.Methods Primary cultured umbilical cord MSCs were identified by flow cytometry,and peripheral blood CD4+ T cells were sorted in pSS patients.CD4+ T cells were cultured with CD3,CD28 antibody for 72 h to be the activated(control group);the activated CD4+ T cells were co-cultured with MSCs for 72 h(MSCs group) or MSCs were pre-stimulated with interferon-γ (IFN-γ),then the activated CD4+ T cells were co-cultured with pre-stimulated MSCs for 72 h (pre-stimulated group).The suspension of CD4+ T cells were collected and counted.PGE2,HGF and IDO in the supernatants were detected by ELISA.Mean in groups were compared using ANOVA,and multiple comparisons were used with LSD method.Results The concentrations of PGE2 in the supernataut of the control group,MSCs group and pre-stimulated group were (111 ±4) pg/ml,(2 814±6) pg/ml and (2 716±8) pg/ml (F=167 292.12,P＜0.01) respectively.The concentrations of HGF in the above groups were (597±9) pg/ml,(383±9) pg/ml and (727±12) pg/ml(F=878.61,P＜0.01) respectively.The concentrations of IDO in the above groups were (143±4) pg/ml,(835±5) pg/ml and (588±3) pg/ml (F=21 104.41,P＜0.01) respectively.Compared with the control group,levels of PGE2 significantly increased in the MSCs group and the pre-stimulated group that CD4+ T cells were co-cultured with MSCs (t=509.88,P＜0.01 and t=491.48,P＜0.01),and levels of IDO also significantly increased (t=202.69,P＜0.01 and t=130.39,P＜0.01),while the activation and proliferation of CD4+T cells were inhibited (t=-16.20,P＜0.01 and t=-31.48,P＜0.01).Compared with MSCs group,levels of PGE2 and IDO significantly decreased in pre-stimulated group (t=-18.40,P＜0.01 and t=-72.30,P＜0.01),and levels of HGF significantly increased in pre-stimulated group(t=41.51,P＜0.01),while the activation and proliferation of CD4+ T cells were further inhibited (t=-15.28,P＜0.01).Conclusion MSCs can inhibit the activation and proliferation of CD4+ T cells in pSS in vitro.The suppressive effect of MSCs may be achieved by promoting secretion of cytokines such as PGE2,HGF and IDO.HGF plays a more important role in the suppressive effect of MSCs pre-stimulated with IFN-γ.Too much PGE2 or IDO propably results in negative feedback regulation of MSCs.

Objective This study aims to understand the characteristics of the time sequence of ICR mouse first mandibular molar tooth germ development through dynamic observation.Methods Tooth germ of Embryos (E11.5,E12.5,E13.5,E14.5,E15.5,E16.5,E17.5 and E18.5) and postnatal (PN1,PN2) mice were obtained.The heads (E11.5-E15.5) and mandibles (E16.5-PN2) of mice were dissected,fixed and embedded for serial sections and HE staining.All the results were assessed under light microscopy.Results The tooth germ underwent various development stages including the bud,cap and bell stages.Mouse odontogenesis was initiated at E11.5.Proliferation of oral epithelium formed the bud stage at E13.5.Then the cap stage was observed at E14.5-E15.5 and the bell stage was appeared beginning from E16.5.The pre-dentin was observed at PN1,as well as the dentin at PN2.Conclusions Establishing the regular development pattern of the first mandibular molar of ICR mice will provide a reliable basis for the future use in the specific tooth germ developmental research.