Reappraisal of the Mawangdui Han Tomb Cadaver Thirty Years After Its Unearthing.

The Mawangdui tomb No.1 cadaver, a female corpse from the Western Han Dynasty, was unearthed in 1972. Forensic examination at the time of discovery indicated fairly remarkable presence of bodily constituents at the anatomical, histological, and molecular levels. The cadaver was preserved in a formalin-based fixative afterward, and maintained in the Hunan Museum. To better protect this rare human corpse, a reappraisal of the status of preservation was carried out using noninvasive approaches, including X-ray radiography, gross anatomical examination, and histological, microbiological, and molecular analyses of sampled tissues. The cadaver remained essentially intact from a gross anatomical perspective, with radiography of the skeletal system and arterial contrast filling appeared comparable with the original documentation. The light microscopic features of the skin, cartilage, and skeletal muscle remained detectable, as were the stratified ultrastructure of the collagen and muscle fibers. The levels of nitrogen and amino acidic elements appeared elevated in the cadaver and liver preservation fixatives, with a higher calcium and phosphate concentration in the former. These findings suggest that there existed a certain degree of macromolecule degradation and bone decalcification in the cadaver, likely irrelevant to biological decomposition. The reappraisal also led to the implementation of stronger scientific measures to better protect the cadaver through a renovated Museum-University coadministrative management agreement.

Inhibiting the proliferation and metastasis of hilar cholangiocarcinoma cells by blocking the expression of vascular endothelial growth factor with small interfering RNA.

The aim of the present study was to investigate whether the proliferation and metastasis of hilar cholangiocarcinoma cells can be suppressed and whether apoptosis can be induced by small interfering RNA (siRNA) repression of vascular endothelial growth factor (VEGF). siRNA sequences targeting the VEGF gene were designed and the human hilar cholangiocarcinoma QBC939, HCCC-9810 and RBE cell lines were transfected with VEGF-siRNA plasmids for 48 h. Reverse transcription-quantitative polymerase chain reaction and western blotting measured the levels of VEGF-A, VEGF-C and matrix metalloproteinase 2 (MMP2) mRNA expression and protein content. The cell invasion potential was evaluated using the Transwell invasion and migration assay and the MTT assay was employed to detect the proliferation of hilar cholangiocarcinoma cells. Flow cytometry was used to quantify cell apoptosis and necrosis. Following the transfection of VEGF-siRNA, a significant reduction of mRNA and protein levels of VEGF-A, VEGF-C and MMP2 was observed in the hilar cholangiocarcinoma cells. The invasion, migration and proliferation of tumor cells were also notably decreased. The rate of tumor cell apoptosis was increased in the VEGF-siRNA group (15.42%) compared with the non-siRNA control (2.22%) and the negative control (2.71%) groups. It was concluded that blocking the expression of VEGF via VEGF-siRNA effectively inhibited the invasion, migration and proliferation, and induced apoptosis in hilar cholangiocarcinoma cells. These observations suggested that targeting VEGF with RNAi may be an effective therapeutic strategy for treating hilar cholangiocarcinoma.

Silencing of CXCR4 inhibits tumor cell proliferation and neural invasion in human hilar cholangiocarcinoma.

BACKGROUND/AIMS: To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. METHODS: An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. RESULTS: The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation. CONCLUSIONS: CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

In vitro neuraotropic growth of cholangiocarcinoma: an experimental study.

OBJECTIVE: Perineural invasion of cholangiocarcinoma happens in the early stage of the disease but is often not recognized until its later stages. Research about the behaviour and mechanism of perineural invasion by cholangiocarcinoma is urgently needed for a useful new model. The aim of this work is to establish a novel model to address the problem. DESIGN: Neural cells and cholangiocarcinoma cells were co-cultured to mimic the neurotropic invasion of cholangiocarcinoma. SETTING: Human embryonic stem cells were induced to form neural cells by glial cell-derived neurotropic factor and retinoic acid; neural cells and cholangiocarcinoma cells were co-cultured in Transwell chamber. PARTICIPANTS: Human embryonic stem cells and cholangiocarcinoma cells were applied. MAIN OUTCOME MEASURES: Paired t-test was used to compare the counts of penetrating cholangiocarcinoma cells in co-culture and control group. RESULTS: Formation of neurospheres and neural-like cells were observed following induction at 24 and 48 h, respectively; synapses were viewed to protrude from neural-like cell bodies after incubation for 96 h. Forty-eight hours after incubation, immunocytochemical staining of the cells showed that synaptophysin and glial fibrillary acidic protein were expressed in the neuron-like cells and gliocytes-like cells, respectively. The cholangiocarcinoma cells that had penetrated through the Matrigel/polyethylene terephthalate membrane from the upper chamber to the lower chamber of the Transwell in the co-culture group were significantly more numerous than those in the control group (68 ± 8.3/field versus 46 ± 5.7/field, P < 0.05). CONCLUSION: The novel model is a valuable tool to study the perineural invasion of cholangiocarcinoma.

A Disintegrin and Metalloprotease with Thrombospondin Motif 2 May Contribute to Cirrhosis in Humans through the Transforming Growth Factor-ß/SMAD Pathway.

BACKGROUND/AIMS: We aimed to investigate the correlation between a disintegrin and metalloprotease with thrombospondin motif 2 (ADAMTS-2) and transforming growth factor-ß1 (TGF-ß1) in clinical human cirrhotic tissues. METHODS: The liver tissues of 24 patients (16 cases with cirrhotic portal hypertension as the cirrhosis group and eight cases with healthy livers as the normal group) were collected. Immunohistochemistry and Western blots were performed to evaluate the protein expression levels of ADAMTS-2 and TGF-ß1. Western blots for other key mediators of cirrhotic progression, including SMAD2, SMAD3, TGF-ß receptor II (TGFßRII), matrix metalloproteinases 2 (MMP2), and tissue inhibitor of matrix metalloproteinases 2 (TIMP2), were also performed. RESULTS: Cirrhotic tissues showed higher percentages of collagen. The protein expression levels of ADAMTS-2 and TGF-ß1 were significantly higher in the cirrhotic group as compared to the matched normal group (p<0.05), and there was a positive correlation between these two proteins (r=0.862, p<0.01). The protein expressions of MMP2, TIMP2, and TGFßRII, as well as the phosphorylated forms of SMAD2 and SMAD3, were significant higher in the cirrhotic group (p<0.01 or p<0.05). CONCLUSIONS: These findings suggested that ADAMTS-2 and TGF-ß1 may play important roles in the pathogenesis of human cirrhosis; specifically, TGF-ß1 may induce the expression of ADAMTS-2 through the TGFß/SMAD pathway.

A standard approach to expose the recurrent laryngeal nerve during endoscopic thyroidectomy.

PURPOSE: Exposing the recurrent laryngeal nerve (RLN) during all types of thyroid surgery is essential to protect this nerve. Endoscopic thyroidectomy (ET) has gained acceptance from both patients and physicians, in part due to the cosmetic benefits. Therefore, the avoidance of intraoperative RLN impairment during ET is of critical significance. We have developed a standard approach to expose the RLN during ET that prevents RNL impairment. PATIENTS AND METHODS: ET was performed in 120 consecutive patients with thyroid disease. In order to develop a standard procedure that protects the RLN, several steps that differed from the traditional open procedure were introduced. First, the thyroid gland was freed from the isthmus instead of the superior pole. Then, the inferior pole of the thyroid gland was meticulously freed, and the lateral side of the thyroid gland was freed followed by the superior pole. At this point, the RLN was easily visualized in the tracheoesophageal groove. The thyroidectomy was then performed simultaneously with exposure of the RLN from the inferior to superior aspects. All RLNs were exposed when hemithyroidectomies, subtotal thyroidectomies, or total thyroidectomies were performed. The operative time and parathyroid hormone (PTH) and calcium levels were recorded prospectively and analyzed. RESULTS: Using this method, all RLNs were easily exposed within 15 minutes. Only one case of transient RLN palsy occurred due to accidental contact of the harmonic scalpel to the nerve. Postoperative hypocalcemia occurred in 23 cases (19.2%), and the PTH level decreased significantly in 33 cases (27.5%). The PTH levels returned to normal within 3 months. CONCLUSION: Use of the described approach to expose and protect the RLN when performing ET is safe and feasible.

[The effect of sevoflurane inhalation anesthesia only and propofol total intravenous anesthesia on perioperative cytokine balance in lung cancer patients].

AIM: To investigate the effects of sevoflurane inhalation anesthesia only and propofol total intravenous anesthesia on perioperative cytokine balance in lung cancer patients. METHODS: ASA I or II patients undergoing lobectomy for lung cancer were randomly divided into two groups with 45 cases each. In group A, patients received g sevoflurane inhalation anesthesia only and patients in group B received propofol total intravenous anesthesia. The cervical venous blood samples were obtained at the following time points: before induetion of anesthesia(T0), before the start of one-lung ventilation(T1), before the end of one-lung ventilation(T2), after closed chest surgery(T3), after 24 h (T4) . The serum concentrations of IL-6, IL-8 and IL-10 were measured by ELISA. RESULTS: (1) In both groups the concentration of IL-6 increased at T1 and kept raising to a high level at T4 which showed significant differences with that of pre-operation(P < 0.05).Compared between the groups, the concentration of IL-6 at T1 and T2 in group B was lower than that of group A(P < 0.05). (2) In both groups the concentration of IL-8 kept at T1 and T3 which were significant with that of pre-operation(P < 0.01). The concentration of IL-8 decreased apparantly at T4 in both groups, it was significant with that of pre-operation though(P < 0.01).Compared between the groups, the concentration of IL-8 at T1, T2 and T3 in group B were all lower than that of group A (P < 0.05). (3) In both groups the concentration of IL-10 increased at T1 which was significant with that of pre-operation(P < 0.05)and kept at T2 and T3. It dropped somehow at T4 but still maintained at a high level.Compared between the groups, the concentration of IL-10 in group B at T1, T2, T3 and T4 was singicantly higher than that of group A(P < 0.05). CONCLUSION: Propofol causes less inflammatory mediator release and can also modulate the balance of cytokines. It is a better anesthetic for lung cancer than sevoflurane.

Fibro-C-Index: comprehensive, morphology-based quantification of liver fibrosis using second harmonic generation and two-photon microscopy.

We develop a standardized, fully automated, quantification system for liver fibrosis assessment using second harmonic generation microscopy and a morphology-based quantification algorithm. Liver fibrosis is associated with an abnormal increase in collagen as a result of chronic liver diseases. Histopathological scoring is the most commonly used method for liver fibrosis assessment, where a liver biopsy is stained and scored by experienced pathologists. Due to the intrinsic limited sensitivity and operator-dependent variations, there exist high inter- and intraobserver discrepancies. We validate our quantification system, Fibro-C-Index, with a comprehensive animal study and demonstrate its potential application in clinical diagnosis to reduce inter- and intraobserver discrepancies.

Tumor-enhancing activity of Basigin expression in gallbladder carcinoma and its prognostic role.

Basigin (EMMPRIN/CD147) is a multifunctional membrane glycoprotein that is overexpressed in many solid tumors and is involved in tumor invasion and angiogenesis. The main purpose of this study was to investigate the tumor-enhancing activity of Basigin in a gallbladder carcinoma (GC) cell line and in primary GC tissues. A system that blocks Basigin in the human primary GC cell line GBC-SD was developed using RNA interference. GBC-SD cells were transfected with the small interfering RNA that target Basigin, then the proliferative, invasive and migration activities of the cells were assayed in vitro. Additionally, tissue samples from 98 patients with GC and 26 patients with chronic cholecystitis were stained with anti-Basigin antibody for immunohistochemical analysis. Furthermore, the association of Basigin expression with the clinicopathological characteristics and prognosis of the patients was analyzed. siRNA-treated GBC-SD cells exhibited significantly decreased growth ability, invasion and migration capacities compared to control cells in vitro. Moreover, clinicopathological analysis demonstrated that the intensity of Basigin staining in cancerous tissue was significantly associated with the histological type (p=0.02), distant metastasis (p<0.01) and Nevin stage (p<0.01) of GC. A proportional hazards model revealed the survival rate of patients with stronger Basigin expression to be the lowest (p<0.01). These results suggest that Basigin is a prognostic marker and potential therapeutic target for patients with GC.

[Expression and clinical significance of highly expressed protein in cancer (Hec 1) in human primary gallbladder carcinoma].

AIM: To investigate the relationship between the expression of highly expressed protein in cancer (Hec 1) and infiltration, metastasis and prognosis of human primary gallbladder carcinoma (PGC). METHODS: The expression of Hec 1 in was detected 108 patients with PGC by SABC immunohistochemistry with 15 cases of chronic cholecystitis as control. Then, a 5 year follow-up was carnedout in 96 out of 108 patients to analyze the correlation between Hec 1 and prognosis of the patients. RESULTS: The clinical pathological characteristics of PGC and clinical outcome of the patients were associated with the expression of Hec 1. Hec 1 was highhy expressed in cancer tissues with lymph node metastasis and poor differentiation. Especially, a statistical correlation was found with more advanced Nevin stages of PGC (P < 0.05). Moreover, the 5-year survival rate of the patients with PGC whose expression of Hec 1 was positive was significantly lower than that of the patients without Hec 1 expression (P < 0.01). CONCLUSION: Hec 1 may be associated with the development, infiltration and metastasis of PGC. The combination of Hec 1 expression in cancer tissues with clinical staging may faliliate the auurate predication of patients' prognosis.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture.

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

[Expression of Ang2 and Tie2 and their relation with the angiogenesis of hepatocellular carcinoma in rats].

OBJECTIVE: To investigate the relationship between the expression of Ang2, Tie2 and the angiogenesis of hepatocellular carcinoma in rats. METHODS: Thirty-eight healthy male rats were randomly divided into 3 groups: 5 rats in the control group; 25 rats in the experimental group were equally divided into 5-day, 10-day, 15-day, 20-day, and 25-day groups; the other 8 rats were used as the supplement of the experimental group. An allogenic transplanted rat model of CBRH-7919 hepatocellular carcinoma in situ was established by immunosuppression. The expressions of Ang2 and Tie2 were detected by immunohistochemical staining in cancerous tissues of different developmental stages and liver tissues of the control group. At the same time, microvessel density was determined by anti-CD31 immunohistochemical staining. RESULTS: CBRH-7919 hepatocellular carcinoma models were successfully set up in 24 rats. The expression level of Ang2 and Tie2 in cancerous tissues was much higher than that of liver tissues of the control group (P <0.05). The overexpression of Ang2 was pristine and continuous in different developmental stages. The expressions of Ang2 and Tie2 positively correlated with microvessal density in hepatocellular carcinoma (P<0.05). CONCLUSION: The up-regulation of Ang2 and Tie2 may play important roles in the angiogenesis of hepatocellular carcinoma. Ang2 may participate in the start of angiogenesis of hepatocellular carcinoma.

[Surgical treatment of 2465 primary hepatolithiasis cases].

OBJECTIVE: To summarize the experience of surgical treatment of primary hepatolithiasis. METHODS: To analyze the clinical data, operation choice, postoperative complications of 2465 cases of primary hepatolithiasis retrospectively. RESULTS: Of the patients, 2034 received external drainage (82.5%) and 431 received internal drainage (17.5%) and 586 were performed adjunctive partial hepatectomy (23.8%). The postoperative complications were found in 211 cases (8.6%) and 17 cases (0.7%) died after the operation. One thousand seven hundred and sixty-seven cases (71.7%) have been followed up for 2 to 25 years, among them therapeutic effect of 1518 cases (85.9%) was excellent or good, 315 cases (17.8%) had residual stone and 115 cases (6.5%) recurred. CONCLUSIONS: It could decrease the incidence rate of complications, residual stone and recurrence in the patients with hepatolithiasis after surgical therapy to pinpoint the situs of the lithiasis and biliary stricture and managed properly before the surgery.

[Effect of Bcl-2 antisense phosphorothioate oligodeoxynucleotides on bile duct carcinoma cell line QBC939].

OBJECTIVE: To investigate the effect of Bcl-2 antisense oligodeoxynucleotides (ASODN) on the cell proliferation and Bcl-2 expression in bile duct carcinoma cell line QBC939. METHODS: Bcl-2 ASODN and control sequence were transfected into cell line QBC939 by Lipofectamine 2000. The changes of Bcl-2 protein were detected by Western blot. The survival rate and colony formation rate of QBC939 cells incubating with Bcl-2 ASODN were evaluated by trypan blue staining assay and colony forming test. RESULTS: The densitometric analysis of Gel-photograph showed that the level of Bcl-2 protein expression in the ASODN transfected group was significantly lower than that in the controls (P < 0.01). Both trypan blue staining assay and colony forming test demonstrated that Bcl-2 ASODN could partially inhibit the growth of QBC939 cells. After incubating with Bcl-2 ASODN, the survival rate and colony formation rate of QBC939 cells were significantly lower than those of the controls (P < 0.05). CONCLUSION: Bcl-2 ASODN inhibits the cell proliferation in bile duct carcinoma cell line QBC939 by blocking the expression of bcl-2 gene.

Diagnostic and surgical therapeutic features of extrahepatic bile duct carcinoma without jaundice.

AIM: To analyze the diagnostic and therapeutic features of extrahepatic bile duct carcinoma (EBDC) without jaundice. METHODS: Between 1985 and 1999, 101 patients underwent surgery for EBDC in Xiangya Hospital. These patients were divided into two groups: 84 jaundiced patients and 17 non-jaundiced patients according to preoperative serum total bilirubin levels. The clinical manifestations, laboratory findings, location, pathology and surgical resectability of the tumors were compared between the two groups. RESULTS: The laboratory parameters such as hemoglobin, serum albumin ALB, AKP, gamma-GT, and sonography appearance were similar between the two groups, and there was no significant difference in tumor location, pathological type and resectability. However, the number of non-jaundiced patients associated with chololithiasis was significantly greater than that of jaundiced patients (P = 0.008). CONCLUSION: The presence of jaundice is not a reliable criterion for the prediction of the resectability and the extent of tumor progression in extrahepatic bile duct carcinoma. Decreased levels of blood hemoglobin and serum albumin, elevated levels of AKP and gamma-GT, and /or abnormal sonography may be suggestive. Biopsy of a stenotic or thickened bile duct is strongly recommended for a correct diagnosis before the appearance of jaundice.

[Androgen receptor and SMRT].

OBJECTIVE: To explore the interaction between androgen receptor (AR) and silencing mediator for retinoid and thyroid hormone receptor (SMRT) and their interaction site. Methods We recombined and constructed AR, SMRT gene and gene fragments, in vitro translated 35S fusion proteins to investigate the relationship between AR and SMRT using transient transfection, mammalian two-hybrid test, GST pull-down assay, and indirect immunofluorescence staining. Results AR possessed an intrinsic transcriptional repression activity and AR interacted directly with SMRT. One interactive surface on AR was mapped to the ligand-binding domain (LBD), and the presence of DNA binding domain enhanced this interaction. The binding surface on SMRT was mapped to the carboxyl-terminal nuclear receptor interacting domain (ID), and mutation of the LXXXIXXXI/L corepressor motif within this domain interferred with the interaction. CONCLUSION: LBD domain on the AR can interact with ID2 motif on the SMRT.