Characterization Of A Near-infrared Fluorescent Dcpo-tagged Glucose Analogue For Cancer Imaging

Part1 Characterization of a near-infrared fluorescent DCPO-tagged glucose analogue for cancer imagingBackground:It is universally acknowledged that cancer is the major cause of death in the world because of its complicated pathology and difficulty for early diagnosis.It has been identified that early detection can support important strategies for cancer treatment.One of the hallmarks of cancer is the Warburg effect,which is characterized as the high rates of aerobic glycolysis.The glycolytic rates of the rapidly proliferating cancer cells can be up to 200 times greater than normal cells.Recent years,many researchers had paid lots of efforts to conjugate fluorescent dyes with glucose to synthesize glucose analogues which can be uptaken by cells.We can differentiate cancer cells from non-transformed tissues based on the absorption difference of dyes.But several drawbacks of the traditional fluorescent dyes hinder its application for low sensitivity,poor penetration and fast photobleaching.Near-infrared(NIR)dyes have good characteristics,including deep tissue penetration,minimum photo damage to biological samples,and minimum interference from background in living systems after reconstructed in the structure.And they can be absorbed into cells through GLUT-1 of the membrane when conjugated with glucose.Therefore,monitoring cellular glucose consumption is an ideal way to differentiate cancer tissues from non-transformed tissues.And this might be applied in cancer cell bioimaging and bioassay in cancer cells.Purpose:To study the absorption difference of NIR dyes of N2 between cancer cells and non-tumor tissues,the kinetic properties of the cellular uptake and might mechanism.And all this can supply detail parameters for practical application.Methods:1.Culture and collect four kinds cells including HL-7702,Hep G2,GES-1,NCI-N87.Using Western Blotting to detect GLUT-1.2.Using CCK-8 method detecting cellular survival rate after specified concentration of N2 added into medium 6h,24 h respectively.3.Taking fluorescent images after N2 dyes(6,24 ?M)administrated into the four kinds of cell medium 3h.4.Incubating the four kinds of cells with N2 dyes(6,24 ?M)3h,then collecting cells with trypsin to make flow cytometry analysis.5.Incubating HL-7702,Hep G2 cells with N2 dyes(12 ?M)different time respectively,then collecting cells with trypsin to make flow cytometry analysis.6.Hep G2 cells were simultaneously treated with glucose(200 m M)and N2(12 ?M)for 3h,or pretreated with phloretin(200 ?M)for 1 h and subsequently treated with N2(12 ?M)for 3 h.After washed with ice-old PBS for three times,cells were observed under microscope.Preparation for quantitative determination is the same way with above-mentioned,collecting cells with trypsin and then observed with flow cytometry.Result:1.High level of GLUT-1 was observed in hepatic carcinoma cells Hep G2 and gastric cancer cells NCI-N87,while non-detectable band was detected in normal liver cells HL-7702 and normal gastric epithelial cells GES-1.2.N2 did not cause any toxicity to cancer or normal cells.3.Fluorescent images intensity of N2 in Hep G2,NCI-N87 were higher than that HL-7702,GES-1.4.The fluorescence intensities of N2 in Hep G2,NCI-N87 were higher than that in HL-7702,GES-1.5.No increased uptake of N2 was observed in HL-7702 cells during incubation period except a very low passive transport.While in Hep G2 cells,the uptake was increasing in a rapid way with the time and reached the stable peak level at 120 min.6.D-glucose is the natural substrate of GLUT-1 and compete the uptake of N2 dye.As an effective and specific inhibitor of GLUT-1,it can reduce the absorption of N2 obviously.Conclusion:We have confirmed that N2 is transported into cells through GLUT-1,and illustrated the kinetic properties in cancer cells and non-tumor tissues.Compared with traditional NIR dyes,the novel fluorescent dye N2 has several good characteristics.And this can be applied to monitor glucose consumption to differentiate cancer cells and cancer diagnosis.Moreover,it could also be used for the discovery of glucose-uptake regulating agents for obesity or diabetes treatment.Part2 Establishment of a cancer cachexia model on spleen-deficiency mouseBackground:Cachexia is a syndrome with high morbidity,which is happened in the advanced cancer phase.And 20% cancer patients died for it,there are still no effective and specific drugs for cancer cachexia treatment.However,our own country traditional medicine has been identified with value for new drug development by modern pharmacology;and the symptomatic classification of traditional Chinese medicine has been confirmed an ideal strategy to diagnose diseases after many years of clinical practice.But,when the application of traditional Chinese medicine for cancer cachexia in the present stage is still remained on the trail stage based on the experience of clinical traditional medicine.We can not develop novel drugs for cachexia without scientific and reasonable animal experiment.It must be very valuable to establish an ideal model to simulate clinical cancer cachexia and to develop effective drugs for cachexia.In our study,we will induce spleen-deficiency mouse with compound factors(purgation with folium sennae decoction,spleen-injury with overstrain,diet disorder)to observe the occurrence and development of pathology,and this would be helpful to illustrate mechanisms of cancer cachexia.Purpose:The aim of this experiment is to establish a standard cachexia model combined on spleen-deficiency and cancer.Firstly,we united three different factors including purgation with folium sennae decoction,spleen-injury with overstrain and diet disorder to induce spleen-deficiency mouse.Secondly,C26 cell lines were inoculated to induce cancer cachexia.We can analysis the disease characterizations of the animal through this study and to seek the optimum model which can be used to simulate clinical diseases better.Methods:Spleen-deficiency was induced through united three factors including purgation with folium sennae decoction,spleen-injury with overstrain and diet disorder.Folium sennae decoction(1 g/m L)was administrated by IP at 8:00 am everyday,the dosage increased to 0.7 m L from seventeenth day;Overstrain was conducted through forced swimming at 1:00 pm everyday,the criterion exhaustion of mouse including festination,uncoordinated movement and even sinked,twice a day and interval ten minutes;Food was free achieved half-day but water is always abundant.Model for spleen-deficiency mouse: C26 cells(0.1 m L,1x106)were incubated into the subcutaneous on right subaxillary,the whole experiment should be accomplished in one hour.Result:1.Inducement of spleen-deficiency syndromeIn our study,united three factors have induced mouse with lots of symptoms containing two respects.Body signs change,such as spirits atrophy,loose stool,lazy and body weight and temperature declined;The content of gastrin,spleen weight and index,thymus weight and index are both decreased compared with control group.We have successfully induced spleen-deficiency mouse model.2.Inducement of cancer cachexia for spleen-deficiency mouseIn the whole experimental period,the body weight and temperature of control group are higher than spleen-deficiency and spleen-deficiency cachexia group obviously.And the spleen-deficiency cachexia group is much lower than spleen-deficiency group with significant statistics.In the advanced phase of cachexia,both body weight and temperature of spleen-deficiency group and spleen-deficiency cachexia group declined obviously.In our study,weight of gastrocnemius muscle(GW),soleus,extensor digitorum longus(EDL)and tibialis anterior(TA)of spleen-deficiency cachexia group is much lower than cancer cachexia group with significant statistics.The change of epididymal fat mass is same with gastrocnemius muscle.For spleen,there is enormous difference in mass and index between spleen-deficiency cachexia and cancer cachexia.Thymus weight and index of spleen-deficiency cachexia were declined further than cancer cachexia.All the results confirmed that spleen-deficiency cachexia model can simulate the combined characterizations of spleen-deficiency symptoms and cancer cachexia.Conclusion:Together,our study has confirmed that spleen-deficiency cancer cachexia can simulate the combined characterizations of spleen-deficiency and cancer cachexia.Especially in the inducing atrophy of gastrocnemius muscle,soleus,extensor digitorum longus and tibialis anterior and fat lipolysis is better than cancer cachexia.And very close to clinical symptoms.This animal model has supplied a better platform for simulating clinical symptoms of spleen-deficiency cancer cachexia patients and researching related drugs to treat cancer cachexia.