| PART 1Objective:To investigate whether CRC cells of different type are able to uptake Mn2+ in vitro and if the Mn uptake of CRC cells with different metastatic potential is different or not.Material and methods:Human CRC cells SW620,LoVo,DLD-1,HCT15,HCT116,SW480,Caco-2 and normal colon mucosal cell CCD841 CoN were cultured.Transwell test was performed to determine the invasion index.Two cell lines with highest index and two with least index were selected to perform MEMRI in vitro.Before MEMRI MTT was performed to assess the cell viablity after being incubated in 0.1 mM Mn for 60 min.Each cell line was evenly divided into two portions,one portion was incubated in culture medium with 0.1 mM Mn and the other one was cultured in medium without Mn,both for 60 min.After being centrifugated and rinsed for 2 times both cell pellets were undergone MEMRI and T1WI,T1 map were performed.T1 map data was transferred to program in MatLab platform to generate T1 map images and ROI was manually drawn on mapping images to calculate T1 value.Average T1 value shortening was recorded as T1 value in cells without Mn incubation minus that in cells with 0.1 mM Mn incubation.One-way ANOVA was used to compare the differences of cell viability in MTT and T1 value shortening between cells.Results:Cells with highest invasion index were SW620,LoVo,HCT116(22.6±0.7,20.9±0.6,173.0± 0.6);the least were DLD-1,HCT15,SW480(7.5±0.3,8.3±0.2,9.7±0.4);the invasion index of Caco-2 was the intermediate level(12.6±0.5).After incubation the cell viability(%)of SW620,LoVo,DLD-1,HCT15,CCD841 CoN was 96.3 ±0.01,96.3±0.03,96.0±0,01,96.2±0.03,96.1±0.02 respectively,the difference was not significant(p>0.05).All cells have shown enhancement on T1WI with cancer cells more prominent.On T1 map the average T1 value Shortening(msec)of SW620 and LoVo were the greatest(289.33±0.57,268.45±6.87);DLD-1 and HCT15 were intermediate(148.68±3.99,128.60±.96);CCD841 CoN was the lowest(65.12±0.12).The difference between high and low metastatic potential cells as well as normal cell,SW620 and LoVo,HCT15 and DLD-1,CCD841 CoN was significant(p<0.001).Conclusion:SW620,LoVo,DLD-1,HCT15 and normal cell CCD841 CoN all took up Mn in vitro,and the cell viability was not significantly changed after 60 min incubation in 0.1 mM Mn.High metastatic potential cells SW620 and LoVo demonstrated greater average T1 value shortening than that of low metastatic potential cells HCT15 and DLD-1.PART 2Objective:To compare the Ga-and Mn-enhancement appearance of SW620 and DLD-1 subcutaneous xenografts;verify the in vitro MEMRI study results of cells.Material and methods:Subcutaneous xenografts of SW620,LoVo,DLD-1,HCT15 cells were developed in BALB/c nude mice.MRI was performed when tumor size reached 5,10 and 15 mm respectively.T1WI,T1 map and Mn-enhanced T1WI,T1 map were performed.For SW620 and DLD-1 tumors Gd-DTPA enhanced MRI was performed before Mn-enhancement.All tumors were undergone H&E and CD34 staining for histopathological evaluation and microvessel density count.T1 map data was transferred to program on MatLab platform.Enhancement pattern,degree and number of tumors with marked enhancement on Ga-and Mn-enhanced MRI as well as the difference of T1 value shortening between tumors with different malignant potential were compared.Results:Thirty-six tumors were successfully developed and all were undergone Mn-enhanced MRI,of which twenty-one(SW620=5,DLD-1=16)were performed Ga-enhanced MRI.All tumors have shown heterogeneous enhancement pattern both on Ga-and Mn-enhanced MRI T1WI though the number of tumors with marked enhancement and enhancement degree varied.T1 value shortening ?500 msec was considered as marked enhancement.Number of tumors shown markedenhancement on Mn agent was more than that on Ga agent(17/23,1/21),however the statistical analysis was not reasonable due to limited sample size.Average T1 value shortening of 5 mm group tumors was greater than 500 msec on Mn agent and less than 300 msec on Ga agent.The paired t test has shown significant difference(p<0.001),though there was no significance in 10 or 15 mm group.In 5 mm group,the average T1 value shortening of high and low aggressiveness tumors SW620,LoVo and DLD-1,HCT15 was greater than 600 msec and less than 300 msec,respectively.In 10 mm group,the average T1 value shortening of high and low aggressiveness tumors LoVo and DLD-1,HCT15 was greater than 600 msec and less than 350 msec,respectively.One-way ANOVA showed significant difference both in 5 and 10 mm group(p<0.001,p=0.011).No significance was found between SW620 and LoVo as well as DLD-1 and HCT15.All tumors were low-intermediate differentiated adenocarcinoma.Few neovascularity was found on MVD,with highest and lowest value of 20 and 11.4 per field,average value was 14.68.Conclusion:Mn agent was able to markedly enhance tumors with minimum Ga-enhancement and might be suitable to detect small tumors.The average T1 value shortening of high malignant potential Tumors was greater than that of lower counterparts.PART 3Objective:To quantitatively assess MnSOD expression of colon cancer cells and normal cell and observe its relation to malignant potential.To investigate the possible relationship between Mn enhancement manifestation and MnSOD expression of tumor.Material and methods:High metastatic potential cells SW620,HCT116,LoVo and low metastatic potential cells DLD-1,HCT15,SW480 as well as Caco-2 cells with intermediate invasion index were undergone western blot to determine the MnSOD expression level,compared with normal cell CCD841 CoN.One-way ANOVA was used to compare the difference of MnSOD expression level.The correlation between MnSOD expression level and invasion index of all cell lines from part 1 was analyzed.MnSOD expression level was further linked to the average T1 value shortening of SW620,LoVo,DLD-1,HCT15 cells.Results:All cells have expressed higher level of MnSOD in various degree,except that the expression of Caco-2(0.163±0.035)was lower than CCD841 CoN(0.185±0.038).The cells with highest expression were all low metastatic potential cells DLD-1 and HCT15(0.777±0.031,1.045±0.038),though relatively low expression cells included both high,low and intermediate aggressiveness cells LoVo,SW620,HCT116(0.438±0.028,0.255±0.018,0.289±0.028),SW480(0.337±0.025)and Caco-2(0.163±0.035).Compared with normal counterpart the MnSOD expression of DLD-1 and HCT15 was markedly increased(p<0.01,p<0.001).There was no correlation between MnSOD expression level and invasion index(r=-0.204,p=0.627).The expression of LoVo and SW480 increased(p<0.05,p<0.05),expression of SW620 and HCT116 increased but without significance.LoVo and SW620 cells that demonstrated greater T1 value shortening on MEMRI were low MnSOD expression,DLD-land HCT15 cells that demonstrated less T1 value shortening on MEMRI were high MnSOD expression.Conclusion:MnSOD expression of colon cancer cells was not corresponding to the malignant potential.For LoVo,SW620,DLD-1 and HCT15,cells with greater T1 value shortening were low MnSOD expression and cells with less T1 value shortening were high MnSOD expression. |
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