In Vivo Bioluminescence Imaging of Transplanted Mesenchymal Stromal Cells and Their Rejection Mediated by Intrahepatic NK Cells.

PURPOSE: Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant. PROCEDURES: Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells. RESULTS: We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R 2 = 0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death. CONCLUSION: Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

[Clinical application of 320-row computed tomography 4D digital subtraction angiography in hepatocellular carcinoma].

OBJECTIVE: To explore the application value of 320-row computed tomography (CT) 4D digital subtraction angiography (DSA) for hepatocellular carcinoma (HCC). METHODS: A total of 40 HCC patients received 320-row CT contrast scans. The 4D DSA images were obtained on the basis of baseline data. The normal anatomy and anatomical variations of hepatic artery, tumor supplying arteries, tumor vessels, tumor staining were observed by comparing DSA (n = 20). RESULTS: 320-row CT 4D DSA could show 6-7 levels of intrahepatic arterial branch. Normal hepatic artery anatomy was found in 35 cases (87.5%, Michels I type) and variations in 5 cases (12.5%). The diagnose accordance rate was 100% between 4D DSA and DSA in showing the anatomy and variation of hepatic artery. Among them, 320-row CT 4D DSA showed tumor staining (n = 40), tumor vessels (n = 28), tumor supplying arteries (n = 26) and two hepatic supplying arteries (n = 3). The number of tumor supplying arteries observed by 4D DSA (n = 20) was 18 versus 19 by DSA. Compared with DSA, the accurate rate of 4D DSA was 94.7% (18/19) in detecting tumor supplying arteries. CONCLUSION: As a noninvasive vascular examination modality, 320-row CT 4D DSA can accurately visualize normal anatomy and variation of hepatic artery, dynamically display tumor staining and reproducibly delineate the three-dimension relationship between tumor and blood vessels. In consistency with DSA in detection blood supply of HCC, 320-row CT 4D DSA provides a rapid, DSA-like and non-invasive alternative.

[To evaluate the role of OLT on splenomegaly of portal hypertension by the radiological changes of splenic morphology and collaterals].

OBJECTIVE: To explore the effect of orthotopic liver transplantation (OLT) on portal hypertension by observing the radiological changes of splenic volume and collaterals before and after OLT. METHODS: In our hospital 56 patients performing OLT due to cirrhosis, portal hypertension and splenomegaly were classified into five groups according to their following-up time: A (≤3 months), B (>3-6 months), C (>6-12 months), D (>12-24 months), and E (>24 months). Twenty health people were chose as control group (F). The splenic width, thickness, length, volume, diameter of portal and splenic vein and collaterals were measured and observed in every patient of six groups before and after OLT respectively. RESULTS: After OLT, the splenic volume decreased by 25.4%, 27.8%, 21.9%, 25.2%, 27.7% in five groups respectively, which was still larger than the normal group (P<0.05). Gastroesophageal varices in 31 cases (81.6%, 31/36) became normal after OLT. The opened umbilical vein disappeared and the retroperitoneal varices persisted in five cases after OLT. CONCLUSIONS: Splenomegaly and opened collaterals can be relieved by OLT effectively. The splenic volume didn't change obviously until it decreased by 25% in the three months after OLT. Gastroesophageal varices can be removed in most of patients after OLT. The splenomegaly could last paralled with the splenic vein and retroperitoneal varices after OLT. After OLT, correct disposal of splenic and collateral changes could improve the success rate and the long-term treatment effect of OLT.

[Enhanced green fluorescence protein reporter gene labeling of mesenchymal stem cells mediated by lentivirus].

OBJECTIVE: To explore the effect of enhanced green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells (MSC), and whether the EGFP gene expression is permanent and stable. METHODS: MSC were infected with EGFP lentivirus at different virus multiplicity of infection (MOI). EGFP positive rate was measured with fluorescent-activated cell scanning (FACS) analysis, and EGFP expression in MSC was investigated under a fluorescence microscope. Cell viability, proliferation, apoptosis and cell cycle were detected with trypan blue stain, MTT colorimetric assay, Hoechst stain and FACS analysis respectively. To evaluate the stability of EGFP expression, EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2, 4, 8 or 16 weeks, and EGFP positive rate and fluorescence strength were detected with FACS analysis. RESULTS: After infected with EGFP lentivirus (MOI = 20) for 96 h, EGFP positive rate of MSC was 97.39% +/- 0.68%. Cell viability, proliferation, apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected, as compared with control MSC (P > 0.05). When cultured in vitro continuously for 2, 4, 8 or 16 weeks, EGFP positive rates of EGFP-MSC were 97.50% +/- 0.54%, 97.32% +/- 0.51%, 97.39% +/- 0.11%, and 97.48% +/- 0.13% respectively, while EGFP fluorescence strength were 440 +/- 13, 445 +/- 12, 458 +/- 13 and 456 +/- 16 respectively. Both EGFP positive rate and fluorescence strength kept in a stable level. CONCLUSION: EGFP lentivirus can efficiently label MSC and has no significant effect on the biophysical properties of MSC. EGFP gene expression in MSC is permanent and stable. EGFP-MSC can be used for further cell tracing research.

[Magnetic/luminescent quantum dots bifunctional nanoparticles labeling of rat bone mesenchymal stem cells].

OBJECTIVE: To evaluate the dual-labeling efficiency of magnetic and luminescent quantum dots bifunctional nanoparticles to rat bone mesenchymal stem cells (BMSC). METHODS: Rat BMSC were isolated, purified, and expanded. Magnetic/luminescent bifunctional nanoparticles (Fe(3)O(4)@CdTe@SiO(2)), were prepared by using silicon dioxide (SiO(2)) to encapsulate simultaneously Fe(3)O(4) and CdTe quantum dots. BMSC were incubated with the Fe(3)O(4)@CdTe@SiO(2) nanoparticles which iron concentration was 25 microg/ml. Fluorescence microscope was used to detect the fluorescence of the intracellular nanoparticles. The dual-labeled BMSC with various concentration underwent ex vivo MR imaging with T(1)WI, T(2)WI and T(2)(*)WI sequences. To show the intracellular iron of labeled cells, prussian blue stain was performed. Spectrophotometer was used to detect the iron concentration in the cells. RESULTS: Intracellular red fluorescence of Fe(3)O(4)@CdTe@SiO(2) can be observed via fluorescent microscopy. Rat BMSC could be effectively dual-labeled with approximately 90% efficiency. The MR images with T(2)WI and T(2)(*)WI sequences, especially with T(2)(*)WI sequence, showed that the signals of the dual-labeled BMSC were lower than those of the unlabeled cells. Cellular total iron is 14.05 + or - 4.15 pg per cell. Iron containing intracytoplasmic vesicles could be observed with Prussian blue staining. CONCLUSION: Rat BMSC can be dual-labeled successfully with Fe(3)O(4)@CdTe@SiO(2) magnetic/luminescent bifunctional nanoparticles successfully, and might serve as a tool for magnetic resonance imaging and in vivo optical imaging.

MR diffusion-weighed imaging of rabbit liver.

AIM: To study the techniques of MR diffusion-weighed imaging (DWI) for normal rabbit liver. METHODS: After 15 normal New Zealand white rabbits and one New Zealand white rabbit implanted with VX-2 tumor were anesthetized with 3% soluble pentobarbitone, DWI was performed respectively for different b values, repetition times (TR) or thicknesses, when other parameters were the same and magnetic resonance imaging (MRI) was performed respectively, or with different field of views (FOV) or coil when other parameters were the same. The distinction between groups was analyzed by SPSS10.0 with apparent diffusion coefficient (ADC), quality index (QI) or signal-noise ratio (SNR). RESULTS: As b value increased, liver ADC, QI and SNR of DWI became smaller and simultaneously (F = 292.87, 156.1, 88.23, P<0.01). QI of DWI was high, when b value was 10, 50 or 100 respectively, but the distinction between them was insignificant; when b value was 800, QI and SNR of DWI were low. QI and SNR of DWI had no significant difference between TR = 4 000, 6 000 and 8 000. QI of DWI with 2 mm thickness was bigger than that with 5 mm thickness (t = 3.04, P<0.01), but SNR of DWI with 2 mm thickness was significantly smaller (t = -17.86, P<0.01). SNR of MRI with knee joint coil was obviously bigger than that with cranium coil (t = -5.77 (T1WI) or -4.02 (T2WI), P<0.01), but QI of MRI was smaller on the contrary (t = 7.10 (T1WI) or 3.97 (T2WI), P<0.01). When FOV was enlarged gradually, SNR of MRI increased (F = 85.81 (T1WI) or 221.96 (T2WI), P<0.01), but QI firstly increased, then decreased (F = 68.67 (T1WI) or 69.46 (T2WI), P<0.01) and QI of MRI was the biggest when FOV was 20 cm x 15 cm. CONCLUSION: The scanning technique is very important in DWI of rabbit liver and the overall quality of DWI with b (100 s/mm2), thickness (2 mm), cranium coils and FOV (20 cm x 15 cm) was best in our study, when other parameters were the same.

MR diffusion-weighted imaging of rabbit liver VX-2 tumor.

AIM: To investigate the implanting method of rabbit liver VX-2 tumor and its MR diffusion-weighted imaging (DWI) characteristics. METHODS: Thirty-five New Zealand rabbits were included in the study. VX-2 tumor was implanted subcutaneously in 14 rabbits and intrahepatically in 6 for pre-experiments. VX-2 tumor was implanted intrahepatically in 12 rabbits for experiment and three were used as the control group. DWI, T1- and T2-weighted of MRI were performed periodically in 15 rabbits for experiment before and after implantation. The distinction of VX-2 tumors on DWI was assessed by their apparent diffusion coefficient (ADC) values. The statistical significance was calculated by analysis of variance (ANOVA) of the randomized block design using SPSS10.0 software. RESULTS: The successful rate of subcutaneous implantation of VX-2 tumor was 29% (4/14) while that of intrahepatic implantation of it was 33% (2/6) in the pre-experiment. The successful rate of intrahepatic implantation of VX-2 tumor in the experiment was 83% (10/12) and 15 tumors grew in 10 successfully implanted rabbits. The DWI signal of VX-2 tumor was high and became lower when the b value increased step by step. The signal of VX-2 tumor on the map of ADC was low. When the b value was 100 or 300 s/mm2, the ADC value of normal group and VX-2 tumor group was respectively 2.57+/-0.26, 1.73+/-0.31, 1.87+/-0.25 and 1.57+/-0.23 mm2/s. Their distinction was significant (F = 43.26, P<0.01), the tumor ADC value between b values 100 and 300 s/mm2 was significant (Tukey HSP, P<0.05) and the ADC value between VX-2 tumor and normal liver was also significant (Tukey HSP, P<0.01). VX-2 tumor developed quickly and metastasized early to all body, especially to the lung, liver, lymph nodes of mediastinum, etc. CONCLUSION: The DWI signal of rabbit VX-2 tumor has its characteristics on MR DWI and DWI plays an important role in diagnosing and discovering VX-2 tumor.