Formation of microchimerism in rat small bowel transplantation by splenocyte infusion.

AIM: To investigate the effect of donor splenocyte infusion combined with cyclosporine A (CsA) on rejection of rat small bowel transplantation (SBT). METHODS: Male Sprague-Dawley (SD) rats and female Wistar rats weighing 230-270 g were used as donors and recipients respectively in the study. Heterotopic small bowel transplantation was performed. The rats were divided into three groups: group one receiving allotransplantation (SD rarr Wistar), group two receiving allotransplantation (SD rarr Wistar) + donor splenocyte infusion, group three receiving allotransplantation (SD rarr Wistar) + donor splenocyte infusion + CsA followed by CsA 10 mg/kg per day after transplantation, in which recipient Wistar rats were injected with 2 x 10(8) SD splenocytes 28 d before transplantation, and treated with CsA after transplantation. Finally, the specific DNA fragment of donor Y chromosome was detected in recipient peripheral blood and skin by PCR. The survival time after small bowel transplantation was observed. Gross and histopathological examinations were performed. RESULTS: The survival time after small bowel trans-plantation was 7.1 +/- 1.2 d in group 1, 18.4 +/- 3.6 d in group 2 and 31.5 +/- 3.1 d in group 3. The survival time was significant longer (P < 0.01) in group 3 than in groups 1 and 2. The gross and histopathological examination showed that the rejection degree in group 3 was lower than that in groups 1 and 2. CONCLUSION: Donor splenocyte infusion combined with CsA decreases remarkably the rejection and prolongs the survival time after rat small bowel transplantation.

[The study on the morphology character of blood-spleen barrier].

OBJECTIVE: To study the morphology and functional character of blood-spleen barrier (BSB) and establish the concept of BSB. METHODS: Thirty healthy Wistar rats were studied. Ten rats were injected with 1.5 ml mixed fluid of India ink and physiological saline through the tail vein. Histological changes of the spleen in all animals were observed with light and electron microscopy, including HE, Foot, Masson staining and immunohistochemistry of CD68 and CD34. RESULTS: Most of the carbon particles were within the splenic sinuses in marginal zone but not in the white pulp after 6 h. There was a characteristic distribution of the macrophagocytes, vessel endothelial cell, reticular tissue and collagen fiber in the BSB. CONCLUSIONS: BSB, surrounding the white pulp, is composed of macrophagocytes, marginal-sinus-endothelial cells and their basement membrane, the reticular tissue (reticular cells and reticular fibers) and collagen fibers. The role of BSB is to keep the microenvironment of white pulp stable. It becomes mature while the formation of germinal center of the white pulp. The permeability of BSB changes during its development.

[The effect and mechanism of arsenic trioxide on hepatocellular carcinoma].

OBJECTIVE: To investigate the effect and mechanism of arsenic trioxide on hepatoma cell line BEL-7402 and observe the effect and best administration method of arsenic trioxide on hepatocellular carcinoma patients who were not suitable for operation. METHODS: The cell activity and morphologic changes were studied after being treated with arsenic trioxide in different concentrations. The apoptosis was detected by flow cytometry assay and DNA fragmentation assay. The caspase-3 level of mRNA and protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and fluoro-spectrophotometer. The growth inhibition of implant tumor was observed in nude mice treated with arsenic trioxide in different concentrations. Arsenic trioxide was used in hepatocellular carcinoma patients by intravenous dropping and continuous regional infusion through hepatic artery. RESULTS: The effect of arsenic trioxide on hepatoma cell lines was dependent on the time and concentration obviously. The decrease in cell number was preceded by morphological changes in the treated BEL-7402 cells that were characteristic of apoptosis, including membrane blebbing, shrunken cytoplasm, nuclear condensation and loss of adhesion. Flow cytometry assay showed an arrestment at G(2)/M phase and sub-G(1) cell peak. DNA fragmentation assay showed a marked DNA ladder. The mRNA level of caspase-3 was no change in RT-PCR whereas the protein of caspase-3 was increased after added As(2)O(3) 1 - 36 h. Caspase-3 activity began to increase after 2 h and reached a maximal level after 12 h in a linear fashion. Then, the level of caspase-3 was decreased, but still in a high level. The growth inhibition of implant tumors was obviously in nude mice. The intravenous usage of arsenic trioxide could improve the quality of life with low toxicity in hepatocellular carcinoma patients not suitable for operation. The tumor size decreased in 30 patients and AFP value decreased in 19 patients by continuous regional infusion through hepatic artery. CONCLUSIONS: Arsenic trioxide can obviously inhibit the growth of hepatoma cell line BEL-7402 through inducing hepatoma cell apoptosis. The activation and increase of Caspase-3 is the possible mechanism of apoptosis, and the acting point is in pro-enzyme level. The best result of arsenic trioxide on non-operative patients should be gotten in continuous infusion through hepatic artery.

Expression of cell cycle/growth regulator genes in human hepatocellular carcinoma and adjacent normal liver tissues.

We investigated the gene expression of the cell cycle/growth regulators in hepatocellular carcinoma (HCC) through the usage of Atlas human cancer array membranes, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. Hybridization of cDNA array membrane was performed with alpha-32P-labeled cDNA probes synthesized from RNA, isolated from HCC and adjacent non-cirrhotic normal liver. RT-PCR of 24 paired specimens and Northern blot of 4 paired specimens were used to confirm the expression patterns of the cell cycle/growth regulator genes identified by Atlas array hybridization. Among 79 genes related to cell cycle/growth regulators, transcription factor DP2 (TFDP-2) and E2F-3 were up-regulated, whereas dual-specificity mitogen-activated protein kinase kinase 1 (MAPKK1) and cell division protein kinase 3 (CDK3) were down-regulated in HCC. RT-PCR of TFDP-2 gave result consistent with Atlas human cancer cDNA array findings. Northern blot analysis of TFDP-2 and E2F-3 of 4 paired specimens all showed up-regulation in HCC compared to normal liver tissues. The results obtained from Atlas microarray provided for the first time a liver cancer-specific expression profile, which identified the gene expressions comprehensively and systematically. The findings may lead to better understanding of the mechanism of onset and progression of HCC. The rapid and high-throughout method of profiling gene expression by cDNA array provides an overview of the key factors that may be involved in HCC. Some genes are reported for the first time in HCC.

K-ras gene mutation in the diagnosis of ultrasound guided fine-needle biopsy of pancreatic masses.

AIM: To investigate the utility of K-ras mutation analysis of ultrasound guided fine-needle aspirate biopsy of pancreatic masses. METHODS: Sixty-six ultrasound guided fine-needle biopsies were evaluated by cytology, histology and k-ras mutation. The mutation at codon 12 of the k-ras oncogene was detected by artificial restriction fragment length polymorphisms using Bst NI approach. RESULTS: The presence of malignant cells was reported in 40 of 54 pancreatic carcinomas and K-ras mutations were detected in 45 of the 54 FNABs of pancreatic carcinomas. The sensitivity of cytology and k-ras mutation were 74 % and 83 %, respectively. The specialty of cytology and k-ras mutation were both 100 %. The sensitivity and specialty of k-ras mutation combined with cytology were 83 % and 100 %, respectively. CONCLUSION: High diagnostic accuracy with acceptable discomfort of FNAB make it useful in diagnosis of pancreatic carcinoma. Ultrasound guided fine-needle biopsy is a safe and feasible method for diagnosing pancreatic cancer. Pancreatic carcinoma has the highest K-ras mutation rate among all solid tumors. The mutation rate of k-ras is about 80-100 %. The usage of mutation of codon 12 of k-ras oncogene combined with cytology is a good alternative for evaluation of pancreatic masses.

Liver regional continuous chemotherapy: use of femoral or subclavian artery for percutaneous implantation of catheter-port systems.

AIM: To evaluate the feasibility and safety of the intraarterial chemotherapy of the liver cancer by an interventional method, catheter-port system. METHODS: Thirty-two catheter-port systems were implanted percutaneously via the femoral artery or subclavian artery. Chemotherapies were performed 0-5 d after the implantation of the catheter-port systems. The mean interval between two sequential chemotherapies was 4 wk. The occurrence of side effects of the implantation was examined clinically. RESULTS: Implantation of the catheter-port was successful in all patients. Mean patency period was 210 d. One occlusion (3.1%) of the catheter was observed. Displacement of the catheter was observed in one case (3.1%). One patient rated a hematoma in the chest wall as important. Mild hematoma was reported in 8 cases (25%). In 3 of 32 cases (9.4%), mild pain was reported initially, and dysesthesia was reported in seven (21.9%). No patient rated overall discomfort as mild, severe, or important. CONCLUSION: Percutaneous placement is feasible and safe for liver regional continuous chemotherapy. Compared with surgical placement, the overall complication rate is comparable or less.

Profiling of differentially expressed genes in human gastric carcinoma by cDNA expression array.

AIM: To investigate the expression of cancer related genes in gastric carcinoma (GC) through the use of Atlas Human Cancer Array membranes with 588 well-characterized human genes related to cancer and tumor biology. METHODS: Hybridization of cDNA blotting membrane was performed with (32)P-labeled cDNA probes synthesized from RNA isolated from gastric carcinoma and adjacent noncancerous gastric epithelial tissue. AtlasImage, which is a software specific to array, was used to analyze the result. RESULTS: The differentially expression cell cycle/growth regulator in GC showed a stronger tendency toward cell proliferation with 2.7-fold up-regulation of CK1. The promoter genes of apoptosis were down-regulated, including caspase-8 precursor, caspase-9 and caspase-10. Among the oncogene/tumor suppressor genes, ABL2 was down-regulated. In addition, some genes were up-regulated, including matrix metalloproteinse 2(MMP-2), MMP-16(MT3-MMP), SKY, CD9 and semaphorin V. A number of genes were down-regulated, including neuroendocrine-dlg (NE-dlg), retinoic acid receptor gamma and tumor suppressor DCC colorectal. In general, The expression of the cancer progression genes were up-regulated, while the expression of anti-cancer progression genes were down-regulated. CONCLUSION: Investigation of these genes should help to disclose the molecular mechanism of the onset, progression and prognosis of GC. Several genes are reported herein to be altered in GC for the first time. The quick and high-throughout method of profiling gene expression by cDNA array provides us with an overview of key factors that may involved in GC, and may aid the study of GC carcinogenesis and provide molecular targets for diagnosis and therapy. The precise relationship between the altered genes and gastric carcinogenesis is a matter for further investigation.

Clinical short-term results of radiofrequency ablation in liver cancers.

AIM: To study local therapeutic efficacy, side effects, and complications of radiofrequency ablation (RFA), which is emerging as a new method for the treatment of patients with hepatocellular carcinoma (HCC) with cirrhosis or chronic hepatitis and metastatic liver cancer. METHODS: Thirty-six patients with primary and secondary liver cancers (21 with primary hepatocellular carcinoma, 12 with colorectal cancer liver metastases and 3 with other malignant liver metastases), which were considered not suitable for curative resection, were include in this study. They were treated either with RFA (RITA2000, Mountain View, California, USA) percutaneously (n=20) or intraoperatively (n=16).The procedures were performed using the ultrasound guidance. The quality of RFA were based on monitoring of equipments and subject feeling of the practitioners. Patients treated with RFA was followed according to clinical findings,radiographic images, and tumor markers. RESULTS: Thirty-six patients underwent RFA for 48 nodules. RFA was used to treat an average 1.3 lesions per patient, and the median size of treated lesions was 2.5 cm (range, 0.5-9 cm). The average hospital stay was 5.6 days overall (2.8 days for percutaneous cases and 7.9 days for open operations). Seven patients underwent a second RFA procedure (sequential ablations). Sixteen HCC patients with a high level of alpha fetoprotein (AFP) and 9 colorectal cancer liver metastases patients with a high level of serum carcinoembryonic antigen (CEA) have a great reduction benefited from RFA. Four (11.1 %) patients had complications: one skin burn; one postoperative hemorrhage; one cholecystitis and one hepatic abscess associated with percutaneous ablations of a large lesion. There were 4 deaths: 3 patients died from local and system diseases (1 at 7 month, 1 at 9 month, and 1 at 12 month), 1 patients died from cardiovascular shock, but no RFA-related death. At a median follow-up of 10 months (range, 1-24 months), 6 patients (16.7 %) had recurrences at an RFA site, and 20 patients (56.7 %) remained clinically free of disease. CONCLUSION: RF ablation appears to be an effective, safe, and relatively simple procedure for the treatment of unresectable liver cancers. The rate and severity of complications appear acceptable. However, further study is necessary to assess combination with other therapies, long-term recurrence rates and effect on overall survival.

Integrin gene expression profiles of human hepatocellular carcinoma.

AIM: To investigate gene expression profiles of integrin genes in hepatocellular carcinoma (HCC) through the usage of Atlas Human Cancer Array membranes, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Northern blot. METHODS: Hybridization of cDNA array membrane was performed with alpha(32)P-labeled cDNA probes synthesized from RNA isolated from hepatocellular carcinoma and adjacent non-cirrhotic liver. AtlasImage, which is a software specific to array, was used to analyze the result. RT-PCR of 24 pairs specimen and Northern blot of 4 pairs specimen were used to confirm the expression pattern of some integrin genes identified by Atlas arrays hybridization. RESULTS: Among 588 genes spotted in membrane, 17 genes were related to integrin. Four genes were up-regulated, such as integrin alpha8, beta1, beta7 and beta8 in HCC. Whereas there were no genes down-regulated in HCC. RT-PCR and Northern blot analysis of integrin beta1 gene gave results consistent with cDNA array findings. CONCLUSION: Investigation of these integrin genes should help to disclose the molecular mechanism of the cell adhesion, invasive and metastasis of HCC. A few genes are reported to have changed in HCC for the first time. The quick and high-throughout method of profiling gene expression by cDNA array provides us overview of key factors that may involved in HCC, and may find the clue of the study of HCC metastasis and molecular targets of anti-metastasis therapy. The precise relationship between the altered genes and HCC is a matter of further investigation.

Arterial chemotherapy of 5-fluorouracil and mitomycin C in the treatment of liver metastases of colorectal cancer.

AIM: Regional chemotherapy using hepatic artery catheters is a good method of treating patients with colorectal cancer liver metastases. We investigated the survival of patients with liver metastases from colorectal cancer using 5-fluorouracil (5-FU) and mitomycin C Cthrough implantable hepatic arterial infusion port. METHODS: Seventy-five patients with inoperable liver metastases from colorectal cancer were included between March, 1992 and November, 2001. We placed implantable hepatic arterial catheter (HAC) port by laparotomy. 5-FU, 1 000 mg/ m(2)/d continuous infusion for five days every four weeks, was delivered in the hepatic arterial catheter through the port. Mitomycin C, 30 mg/m(2)/d infusion in the first day every cycle through the port. Response to the treatment was evaluated by serial determinations of plasma CEA and imaging techniques consisting of computerized tomography and sonography of liver. RESULTS: Sixty-eight were performed hepatic artery chemotherapy and fifty-six were followed up among seventy-five HAC patients. Twenty-six patients(46.4 %) have responded and 4 complete remission were achieved. Eight patients (14.3 %) had stable liver metastases. Twenty-two patients (39.3 %) were progressed with increased tumor size and number. Twenty-nine patients(51.8%) had a decreased serum CEA level, while 10 patients (17.9 %) were stable and 17 patients (30.4 %) had an increased serum CEA level. There were no operative death in this series. Complications, which occurred in 18 patients (32.1 %), were as followed: hepatic artery thrombosis in 11, Upper gastric and intestinal bleeding in 3, liver abscess in 1, pocket infection in 1, cholangitis in 1, and hepatic artery pseudo-aneurysm in one patient. CONCLUSION: Combined infusion of 5-FU and mitomycin C by hepatic artery catheter port is an effective treatment for liver metastases from colorectal cancer. The high response and lower complication rates prove the adjuvant treatment of colorectal cancer with this treatment.

Gene expression profiles of hepatoma cell line BEL-7402.

BACKGROUND/AIMS: To investigate the gene expression of cancer related genes in hepatoma cell line BEL-7402 through the usage of Atlas Human Cancer Array with 588 well-characterized human genes related with cancer biology. METHODOLOGY: Hybridization of cDNA membrane was performed with 32P-labeled cDNA probes synthesized from RNA isolated from Human hepatoma cell line BEL-7402 and non-cirrhotic normal liver which was liver transplantation donor. Bioinformatics was used to analyze the result. The reverse transcription polymerase chain reaction of 24 pairs of specimens and northern blot of 4 pairs of specimens were used to confirm the expression pattern of some genes identified by Atlas arrays hybridization. RESULTS: The differential expression cell cycle/growth regulator in hepatoma cell line BEL-7402 showed a stronger tendency toward cell proliferation with up-regulation of E2F-3 and TFDP-2. The anti-apoptotic factors such as Akt-1 were up-regulated. Whereas the promotive genes of apoptosis were down-regulated, such as BAK and caspase-3. Besides this, some genes were up-regulated, such as Integrin beta 8, DNA-PK, CSPCP, cyclin C etc. A number of genes were down-regulated, which included LAR, ABL2, SKY, TDGF1 etc. In general, expression of the cancer progression genes were up-regulated, while expression of anti-cancer progression genes were down-regulated. The results of reverse transcription polymerase chain reaction of 24 pairs of specimens and Northern Blot of 4 pairs of specimens were consistent with the expression pattern of some genes identified by Atals arrays hybridization. CONCLUSIONS: Investigation of gene expression profile of hepatoma cell line BEL-7402 should help to disclose the molecular mechanism of the onset, progression and prognosis of hepatocellular carcinoma. The quick and high-throughout method of profiling gene expression by cDNA array provides us with an overview of key factors that may be involved in hepatocellular carcinoma, and may find the clue to the study of hepatocellular carcinoma carcinogenesis and molecular targets of diagnosis and therapy.