Ameloblastoma is a benign tumor characterized by locally invasive phenotypes,leading to facial bone destruction and a high recurrence rate.However,the mechanisms governing tumor initiation and recurrence are poorly understood.Here,we uncovered cellular landscapes and mechanisms that underlie tumor recurrence in ameloblastoma at single-cell resolution.Our results revealed that ameloblastoma exhibits five tumor subpopulations varying with respect to immune response(IR),bone remodeling(BR),tooth development(TD),epithelial development(ED),and cell cycle(CC)signatures.Of note,we found that CC ameloblastoma cells were endowed with stemness and contributed to tumor recurrence,which was dominated by the EZH2-mediated program.Targeting EZH2 effectively eliminated CC ameloblastoma cells and inhibited tumor growth in ameloblastoma patient-derived organoids.These data described the tumor subpopulation and clarified the identity,function,and regulatory mechanism of CC ameloblastoma cells,providing a potential therapeutic target for ameloblastoma.

Pleomorphic adenoma (PA) is the most common benign tumour in the salivary gland and has high morphological complexity. However, the origin and intratumoral heterogeneity of PA are largely unknown. Here, we constructed a comprehensive atlas of PA at single-cell resolution and showed that PA exhibited five tumour subpopulations, three recapitulating the epithelial states of the normal parotid gland, and two PA-specific epithelial cell (PASE) populations unique to tumours. Then, six subgroups of PASE cells were identified, which varied in epithelium, bone, immune, metabolism, stemness and cell cycle signatures. Moreover, we revealed that CD36⁺ myoepithelial cells were the tumour-initiating cells (TICs) in PA, and were dominated by the PI3K-AKT pathway. Targeting the PI3K-AKT pathway significantly inhibited CD36⁺ myoepithelial cell-derived tumour spheres and the growth of PA organoids. Our results provide new insights into the diversity and origin of PA, offering an important clinical implication for targeting the PI3K-AKT signalling pathway in PA treatment.
Humans ; Adenoma, Pleomorphic/genetics* ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Transcriptome ; Myoepithelioma

Objective:To investigate the value of real-time shear wave elastography (SWE) in the diagnosis of acute testicular torsion.Methods:A total of 30 rabbits were randomly divided into 3 groups including control group (Group S), complete torsion group (Group C) and incomplete torsion group (Group U), and corresponding animal models were built. Rabbits in each group were examined by gray scale and color Doppler ultrasound, SWE and contrast-enhanced ultrasound(CEUS) before and after operation. Mean values of elasticity modulus (Emean) of testicular capsule area, parenehyma testis, spermatic cord torsion section, torsion lower section and contrast-enhanced ultrasound parameters were recorded and then pathological examinations were performed.Results:There was no significant difference for Emean values of each group in all parts before operation( P>0.05). There was no significant difference for Emean value of S group at each period after operation( P>0.05). Emean value of postoperative testicular capsule area in U group increased obviously and rapidly compared with C group, yellow or red "hard ring signs" appeared within four to six hours after operation, there was significant difference of Emean value difference at various periods after operation in group U and C, and at 1 h, 1.5 h, 2 h, 2.5 h, 3 h and 3.5 h postoperatively between group U and C( P<0.05). Postoperative Emean value of parenchyma testis in group C and U increased about 5 kPa, expression of SWE was consistent blue. Postoperative Emean value of spermatic cord torsion section in group C increased more obviously compared with U group, a large patch of red signals appeared within five to six hours after operation, there was significant difference of Emean value at various periods after operation in group C and U, and at each period except instant, 2 h, and 3 h postoperatively between group C and U( P<0.05). Postoperative Emean value in spermatic cord torsion lower section in group U and C increased slowly ( P<0.05), expression of SWE changed from blue to blue green and green from original blue. CEUS showed blood pefusion in testis was "in and out slowly " in group U, pathological feature showed the testicular spermatogenic cells were in disorder with interstitial edema, distinct expansion of microvascular lumen, hyperemia and congestion and lots of erythrocyte leakage. CEUS showed postoperative contrast agent was not filling in testicular in group C, pathological feature showed the testicular spermatogenic cells were in disorder with interstitial edema, slight expansion of microvascular lumen, congestion and part erythrocyte leakage. Conclusions:The image changes of SWE after acute testicular torsion are related with the degree and time of torsion, the changes of testicular capsule area and spermatic cord torsion section are most significant. The use of SWE helps to get accurate hardness information of focus with effective clinical value for the diagnosis testicular torsion.

Aim To investigate the modeling of breast cancer in zebrafish embryos and its related protein expression. Methods 48hpf wild type AB/ TG(Transgenic) zebrafishs were micro-in-jected with breast cancer cell line: MCF-7,T-47D, MDA-MB-231 respectively, the relationship between the number of tumor and model application was investigated, and the number of sub-intestinal veins(SIVs) was detected under confocal microscope, as well as the metastasis of tumor cells in embryos; then the ze-brafish xenografts of MB-231 were co-cultured with tofacitinib/ptk787 for 48 h, optical density(OD) of the cell survival and subintestinal veins(SIVs) were evaluated under confocal micro-scope, and Western blot(WB) analysis was used to test micro-circumstances related protein. Results When the number of in-oculated cells was more than 200 per embryo, xenograft model rate woule be more than 0. 90;MB-231 xenografts showed metas-tasis feature in zebrafish, which could be inhibited by tofacitinib (P < 0. 01), while the number of xenograft MB-231 cells was reduced significantly(P < 0. 01); in another zebrafish xenografts SIVs assay, the tumor could promote the proliferation of SIVs, and 4 mg·L - 1 PTK787 showed inhibiton effect( P < 0. 01). Western blot showed 4d T-47D xenograft zebrafish got more HER2 expression than AB embryos; VEGFa expression in ze-brafish MB-231 model group was higher, and model zebrafish P53 expressi was higher after treated by tofacitinib. Conclusion A zebrafish xenograft model of human brest cancer can be es-tablished, which demonstrates applicability for screening com-pounds in drug discovery studies.

Aim To study the effect of rhein on renal damage induced by aristolochic acid. Methods Ze-brafish model of aristolochic acid nephropathy, genera-ted by treating zebrafish larvae with aristolochic acid for 24 h, was treated with rhein simultaneously . Mor-pholigical changes were observed and the creatinine level in larvae tissue was measured. And mRNA ex-pression levels of inflammatory factor cox2 a and fibrosis factor TGF-β1 in larvae tissue were detected using qPCR. Results Some larvae show periocular edema and circulation system defection e. g. weak heart beat, narrow cardiac vesicle, decreased blood flow and even blockage , with a dose-response relationship after expo-sure to aristolochic acid for 24 h. The creatinine level in larvae tissue of the treated group was significantly higher than that of the control larvae. And the expres-sion levels of cox2 a and TGF-β1 in larvae tissue of the treated group were also significantly increased. Per-centage of abnormal larvae and creatinine level in lar-vae tissue were decreased when treated with rhein sim-ultaneously. And the expression levels of cox2a was down-regulated by rhein compared with the aristolochic acid treated group. But rhein had no effect on TGF-β1 expression. Conclusion To some extent rhein can protect renal from damage induced by aristolochic acid.

Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).

Objective To construct a eukaryotic expression vector in Pichia pastoris containing human fibrinogen gene, in order to achieve high level secretory expression in extracellular.Methods Expression plasmid,pGAPZαA-FGB-FGG-FGA-AOX1,was constructed by inserting the synthesized sequence encoding human fibrinogen(FGA, FGB,FGG) and then introduced into Pichia pastoris SMD1168H by electroporation.Transformants were availably screened by Zeocin resistance,the expression of recombinant protein was identified by SDS-PAGE and Western blot analysis, the protein yield was tested by ELISA assay.After ultrafiltration and purification, the biological activity of protein was detected.Results The crude yield of human fibrinogen in Pichia pastoris supernatant reached 15 mg/L in flask and the biological aggregation activity was determined.Conclusion The human fibrinogen gene was obtained and successfully expressed in Pichia pastoris and the active products were secreted into the medium.

Aim To investigate the anti-angiogenesis and anti-xenograftes of UA in zebrafish larvaes. Meth-ods 24 hour post-fertilization ( hpf ) TG zebrafish was treated with different concentration of UA for 24h, and the number of intersegmental vessels( IVS) were detec-ted under fluorescent microscope respectively;then the models of AB/TG zebrafish xenografts were established by be micro-injected with SMMC-7721 or HT-29 cell at 48hpf respectively, and after cocultured with UA for 48h, optical density (OD) of the SMMC-7721 cell and subintestinal veins ( SIVs) induced by HT-29 were e-valuated under confocal microscope. Results ISV as-say showed that UA could cause IVS missing or disap-perance, inhibition ratio reaching 20. 25% and 26. 65%. UA blocked the spread of SMMC-7721 cells in zebrafish and OD value,and inhibition ratios at three concentrations were 38. 01%, 54. 69%, 61. 88%, re-spectively; in another SIVs assay induced by xeno-grafts, UA at concentration 10 and 15mg·L-1 showed that SIVs were inhibited (P<0. 01) obviously. Con-clusion UA could inhibit the angiogenesis and the growth of SMMC-7721/HT-29 xenografts,and the anti-tumor mechanism may be related with VEGFR2 expres-sion.

BACKGROUND:Tissue engineering provides new ideas and approaches for repair of cartilage defects.
OBJECTIVE:To develop a complete set of solutions for construction of tissue engineered cartilagein vitro, with chondrocytes as seed cels and cross-linked sodium hyaluronate as scaffold materials.
METHODS: New Zealand rabbit articular chondrocytes were isolated, counted, and then cultured and passaged to prepare cellsuspension. Toluidine blue staining, RT-PCR and immunocytochemistry were exerted to evaluate the cultured cels. Chondrocytes were seeded and co-cultured with cross-linked sodium hyaluronate scaffold for 21 days. Then, RNA was isolated for RT-PCR, and frozen sections were prepared for morphological observation and immunohistochemistry study.
RESULTS AND CONCLUSION:The chondrocytes could adhere to the cross-linked sodium hyaluronate scaffold and aggregate, growing between fibers or adhering to the scaffold in a monolayer manner. The transcripts of cartilage specific aggrecan gene and colagen type II alpha 1 gene and cartilage specific protein colagen type II were expressed in cel-scaffold complexes to maintain the phenotype of chondrocytes. Cel-scaffold complexes co-cultured in vitro can form cartilage extracelular matrix, by which tissue engineered cartilage is expected to be obtained.

Objective To retrospectively study the high risk factors for biliary complication (BC) and the application of the Clavien system to classify BC in a large cohorts of subjects undergoing liver transplantations (LT).Methods The clinical data of 181 patients who received LT from Jan.2004 to Dec.2008 were studied.BC was classified using the Clavien system.The risk factors of biliary complication were evaluated by using a binary forward stepwise logistic regression analysis.Results 14.4％ (26/181) recipients developed BC (BC group).In 84.6％ (22/26) patients the BC was above the Clavien Ⅲ b.Regression analysis of BC revealed that the placement of a T tube (P =0.0090,OR=31.177),RIld (P=0.0094,OR＜0.001).RI1w (P=0.0013,OR＞999.999) were significantly associated with the development of BC.Regression analysis of BC above Clavien Ⅲ b revealed that RIld (P=0.0065,OR＜0.001,RI1w (P=0.0022,OR＞999.999) were significantly associated with the development of BC above Clavien Ⅲ b.Conclusions The Clavien classification system was useful to classify BC.The placement of a T tube was an independent risk factor to predict BC,it was not a factor for BC above Clavien Ⅲ b.Hepatic arterial insufficiency (HAI) was an independent risk factor for BC and BC above Clavien Ⅲ b.