[Evaluation of in vivo labeled cytotoxic T lymphocytes and cytokine-induced killer cells migration and distribution of subcutaneous gastric tumor model in nude mice].

OBJECTIVE: To investigate the migration and distribution of CTL (cytotoxic T lymphocyte) and CIK (cytokine-induced killer) cells in gastric tumor model. METHODS: Subcutaneous gastric tumor model was established by BGC-823 cancer cells in nude mice. Both CTL and CIK cells were labeled with 99Tc(m) directly and then inoculated into nude mice with subcutaneous tumor by intravenous injection separately. Three mice of each group were evaluated by single-photon emission computerized tomography (SPECT) at 1 h, 6 h and 24 h post-inoculation. After SPECT imaging, 3 mice in each group were sacrificed and got samples of the tumor, liver, spleen, kidney, lung, intestine, etc. The tissue samples were weighed and radioactivity was determined with a well-type scintillation counter. The accumulation of labeled CTL and CIK cells in tissues were expressed as %ID/g (percentage activity of injection dose per gram of tissue) and T/NT (tumor/non-tumor) values were analyzed. RESULTS: The tracing of both cells in SPECT showed a clear migration path away from the injection point to solid tumor, and can be detected in all organs and tissues such as liver, spleen, kidney, lung and intestine, etc not long after injection. The %ID/g peak values of CTL in organs from the highest to the lowest were as follows: tumor (7.79 +/- 0.46), liver (4.12 +/- 0.51), intestine (2.71 +/- 0.16), kidney (1.44 +/- 0.25), spleen (1.24 +/- 0.12), kidney (1.12 +/- 0.11), and all the T/NTs were above 1. The %ID/g peak values of CIK cells in organs from the highest to the lowest were as follows: liver (6.64 +/- 0.67), tumor (5.47 +/- 0.87), intestine (3.55 +/- 0.23), kidney (2.34 +/- 0.41), spleen (1.45 +/- 0.17), lung (1.27 +/- 0.21), and T/NTs > 1 except for liver. After injection, the %ID/g values of tumor in CTL group were 2.35 +/- 0.28 (1 h), 4.58 +/- 0.52 (6 h) and 7.79 +/- 0.46 (24 h) respectively while the %ID/g values of tumor in CIK group 2.23 +/- 0.46 (1 h), 3.25 +/- 0.70 (6 h) and 5.47 +/- 0.87 (24 h) respectively. At 24 h point, the %ID/g of CTL in tumor was much higher than CIK cells (P < 0.05). CONCLUSION: The definite directional tumor-targeting capacity of CTL and CIK cells in tumor-bearing nude mice is promising.

[Experimental study of implantable engineered liver tissue using type I collagen gel as scaffold].

OBJECTIVE: To construct implantable engineered liver tissue (ELT) using type I collagen gel as scaffold. METHODS: Type I collagen was obtained from the tail of a rat. Hepatocytes were collected from a Sprague-Dawley rat, mixed with liquid type I collagen and Dulbecco's modified Eagle's medium to create hepatocyte/collagen gel construct. The construct was inoculated in a 96-well plate. 0, 3, 5, 7, 9, 11, 13, and 15 days after the inoculation the viability of hepatocytes in vitro was measured by MTT assay. Phase contrast microscopy was used to observe the morphology of the hepatocyte/collagen gel construct. Three SD rats underwent injection of the hepatocyte/collagen gel construct into the subcutaneous space. One week later the implant was taken out. The morphology was conducted by routine H.E. staining and immunohistochemical staining. The morphology and function of hepatocytes was investigated by inverted microscopy, routine H.E. staining and immunohistochemical staining. The constructs were also implanted into subcutaneous space, and the differentiation of hepatocytes and the formation of engineered liver tissue were observed by routine H.E. staining and immunohistochemical staining. RESULTS: Phase contrast microscopy showed that the hepatocytes were distributed evenly in the construct and remained round-shape throughout the in vitro culture. MTT assay demonstrated that the high viability of hepatocytes (87%) was maintained up to 7 days, and then decreased gradually. Albumin, the specific marker of hepatocytes remained positive by immunohistochemical staining after 15-day culture. One week after implantation into subcutaneous space, the implanted hepatocytes retained its hepatocyte-specific morphology, i.e. round shape, large nuclear/cytoplasm ratio as well as binuclear cells, and formed small engineered liver tissue containing blood vessels within and surrounding the tissue. CONCLUSION: A novel approach to construct implantable engineered liver tissue using collagen gel as scaffold for growth and differentiation of hepatocytes has been dev eloped. This technique is an attractive tool for the development of liver tissue engineering.

[Analysis of the early and late gene expression of lipopolysaccharide activated macrophages by cDNA microarray].

OBJECTIVE: To study the early and late changes in mRNA expression in macrophages in response to lipopolysaccharide (LPS) with a cDNA microarray approach using the Clontech Atlas microarray. METHODS: mRNA was isolated from unstimulated control and LPS stimulated murine peritoneal macrophages at 2 hours and 24 hours poststimulation, converted to (33)P radiolabeled cDNA, and hybridized to mouse array membranes. RESULTS: In macrophages being stimulated for 2 hours, 69 out of 1 176 genes were found to differ by over 3-fold compared with the control. Among them 44 genes were up-regulated and 25 genes were down-regulated. In macrophages stimulated for 24 hours, 11 genes were up-regulated and 26 genes were down-regulated compared with the control. Only 8 genes were identified both at 2 hours and at 24 hours poststimulation. The expressions of many genes encoding transcription factor, cytokines, cell signaling modulators and apoptosis associated proteins were found to have changed. Some genes that were not previously linked to this model, such as bric-a-brac (BTB) and cap-n-collar(CNC) homology 1(BACH1), early growth response protein 2 (EGR2), E47 interaction protein 1 (EIP1), Ngfi-A binding protein 2 (NAB2), myeloblastosis oncogene-like protein (MYBL2), neurofibromatosis 1 (NF1), ciliarry neurotropic factor (CNTF) and semaphorin 4A (Sema4A). CONCLUSION: This study has allowed us to identify genes that may potentially be regulated by LPS at early and late phase in macrophages. These may contribute to better understanding of the mechanism underlying LPS or bacteria induced inflammatory and immune response following infection and trauma.

Protective effects of lidocaine injected into the hepatoduodenal ligament on warm ischemia-reperfusion injury to the rat liver.

BACKGROUND: Ischemia-reperfusion (IR) injury to the liver is still a critical and daunting problem in the field of hepatobiliary surgery. Ischemic preconditioning (IP) of the liver serves as an effective approach against IR injury. This study was to develop a novel procedure that could mimic IP, but might be more feasible than IP during surgery. METHODS: Eighty-two SD rats were randomly divided into 5 groups. L group (n = 21): 0.4% lidocaine (10 mg/kg) was injected into the hepatoduodenal ligament 10 minutes before a 40-minute hepatic IR. IP group (n = 16): a 5-minute ischemia was followed by a 10-minute reperfusion prior to a 40-minute hepatic IR. ILR group (n = 15): after a 40-minute ischemia of the liver, 0.4% lidocaine (10 mg/kg) was injected into the hepatoduodenal ligament 10 minutes prior to a 40-minute reperfusion of the liver. IR group (n = 15): the liver of the rat was subjected to a 40-minute IR. Control group (n = 15): 0.9% sodium chloride was injected into the hepatoduodenal ligament without other treatments. The levels of plasma alanine transaminase (ALT) and aspartate transaminase (AST) were determined for each group after treatment. RESULTS: The mean concentrations of ALT and AST were (379.80 +/- 141.69) U/L and (606.05 +/- 220.26) U/L for the L group, (334.64 +/- 141.94) U/L and (625.68 +/- 267.06) U/L for the IP group, (523.36 +/- 170.35) U/L and (765.47 +/- 238.45) U/L for the ILP group, (524.29 +/- 163.59) U/L and (764.63 +/- 246.79) U/L for the IR group, and (150.90 +/- 27.05) U/L and (298.15 +/- 47.68) U/L for the control group (standard error of the mean). CONCLUSION: A significant decrease in ALT and AST levels was observed in the L and IP groups when compared to the ILR and IR groups (P < 0.05), but no significant difference in ALT and AST levels was observed in the L group when compared to the IP group (P > 0.05). These results suggest that pretreatment with lidocaine injected into the hepatoduodenal ligament prior to IR provides effective protection against subsequent IR injury to the liver. The novel approach of blocking innervation with lidocaine mimics hepatic IP, but is more convenient than IP at the time of liver surgery.