| The sarcoma is one of most common primary highly malignant tumors, it origins in the mesenchymic tissue (contracts organization and muscle including knot) the malignant tumor is called "the sarcoma". Occurs in the skin, hypodermic, the periosteum and the long bone both sides.The sarcoma growth is rapid, tumor later period common necrosis, the hemorrhage, cuts the face puttying red, the nature even thin like lives the fish flesh shape, the early time then has a blood line of shift. The osteosarcoma is in the sarcoma the malignant degree is highest, By youth artificial many, take can produce the osteoid organization's spindle-shaped matrix cell as the characteristic, good sends in the growth active dry epiphysis end, especially by the thighbone lower extremity, the shinbone upper extreme and the humerus upper extreme most sees, approximately composes the bone malignant tumor 1/3.The osteosarcoma develops rapidly, the course of an illness is short, starts to grow in the cerebral cortex, may gradually to the medullary cavity of bones development, sometimes outward break through the periosteum, periphery the invasion the soft tissue mesenchymic tissue malignant tumor.At present already proved that, the tumor growth and the shift must rely on the blood vessel production.The volume may organize in the 1-2mm3 following lump body through the osmosis from periphery to obtain the nutrition, maintains own survival.By now the tumor grew extremely slowly, because the tumor further growth had to rely on the newborn blood vessel production, the rich network of bloodvessels provided the sufficient oxygen, the nutrition ingredient and the tumor growth factor for the tumor cell and so on, also was the tumor shift channel.Blood vessel production ability was considered is the tumor growth and the attack symbol.The tumor blood vessel production was from the blood vessel bed which existed has formed the newborn blood capillary the process, degraded, the blood vessel endothelial cell activation, the blood vessel basilar membrane and the extracellular matrix proteinase including the blood capillary basilar membrane degrades, the endothelial cell proliferation and the migration multiplies, the lumen type structure forms steps and so on fusion, blood vessel wall reconstruction, blood stream penetration.This process both influences and so on organism nerve internal secretion factor, and tumor cell and tumor matrix cell expression growth factor regulation.In the tumor blood vessel production process involves to many kinds of presses the blood vessel production factor and between the blood vessel production inhibiting factor adjustment is unbalanced.Not only this process involves presses the blood vessel production factor secretion to increase, moreover the endocardial blood vessel production inhibiting factor has the corresponding reduction.At present has separated and has purified 20 many kinds of blood vessel production factor, the main blood vessel growth promotion factor has blood vessel endothelial cell growth factor (VEGF), the acidity and the alkalinity becomes the ciliary cell growth factor (aFGF, bFGF), the tumor necrosis factor alpha, the transformation growth factor a/p, the chest glucoside phosphorylase, the blood platelet source growth factor, the liver cell growth factor, epidermis growth factor (EGF), blood vessel generator (angiogenin), placenta growth factor (PIGF), the granular cell colony stimulation factor, the blood vessel element, the blood platelet activation factor, the proliferation element, the P material, the lactate, the hyaluric acid fragment and the prostaglandin and so on.The endocardial blood vessel production inhibiting factor mainly includes blood vessel chalone (angiostatin), bast chalone (endostatin), the zymoplasm sensitive protein (TSP-1), metal proteinase inhibitor (TIMP), the blood platelet factor 4(PF4), the interferon alpha (IFN-a), Bai Jiesu-10(IL-10), the soluble VEGF acceptor (sFlt-1), the soluble tyrosine activating enzyme acceptor (sTie-2) and so on.The blood vessel bast growth factor (vascular endothelial growth factor, VEGF) one kind of sequence is highly conservative, highly specificity the factor which presses the blood vessel endothelial cell to grow, widely distributes in organizations and so on human and animal in vivo cerebrum, kidney, liver, spleen, pancreatic gland and skeleton, has the intense cell mitosis function to the endothelial cell, stimulates the blood vessel endothelial cell multiplication and the blood vessel permeability increases, promotion newborn vascularization. VEGF through and blood vessel endothelial cell surface acceptor (vascular endothelial growth factor receptor, VEGFR) specificity union display biology effect.Suppresses VEGF and the VEGFR activeness, may slow down or hinder the osteosarcoma attack and the shift, the osteosarcoma attack and the shift molecular regulation mechanism is the present osteosarcoma molecular biology research hot spot, the research indicated, VEGF and VEGFR make a sneak attack and shift the vital role to the tumor blood vessel and the lymph vessel production and the tumor.The matrix metal proteinase (matrix metallopmteinase, MMP) is a protein hydroltyic enzyme family, may degrade the extracellular matrix (extracellular matrix, ECM) the ingredient.In recent years along with the molecular biology research progress, the scholars to the MMP structure, the function, regulative and the pathology physiological action has conducted more thorough research, MMP in the tumor attack, the shift function has more and more received takes.The metal proteinase organization inhibitor (tissue inhibitor of metalloproteinase, TIMP) is the endocardial MMP inhibitor, it through suppresses the MMP active form and the reactivation process exerts the dual influences.Its C end function area and the MMP other spot union, forms the MMP-TIMP complex, thus blocks MMP and the substrate union, suppresses MMP the activeness, slows down or hinders the osteosarcoma attack and the shift. The osteosarcoma attack and the shift molecular regulation mechanism is the present osteosarcoma molecular biology research hot spot.The research indicated that, MMPs and shifts the vital role to the tumor attackIn 1971 Folkman first proposed about the tumor vascularization as well as the supposition which grows through the anti-vascularization suppression tumor:The tumor growth in thickness achieved when is bigger than 1-2mm3 periphery, the tumor cell may cause the blood vessel endothelial cell to start to multiply, the migration, forms the new blood capillary in the tumour, guaranteed the tumor continues to grow and the shift; May through the suppression tumor blood vessel formation achieve the suppression tumor grows the goal, the suppression tumor blood vessel formation cannot eliminate all tumor cell, but can prevent the tumor cell the growth.And when afterwards experiment confirmed, thus for suppresses the tumor blood vessel production to provide the rationale.In 1989 Ferrarra separated kind of glycoprotein in the cow pituitary gland follicle astral cell nutrient fluid which purified, namely blood vessel bast growth factor, it with tumor cell division, growth, shift all with tumor vascularization related (vascular endothelial growth factor, VEGF), after in 1994 reported Dr.O'Reilly has discovered first endocardial vascularization inhibitor Angiostatin, also in 1997 reported has discovered Endostatin. On September 12,2005, our country independent research and development "reorganizes the human blood vessel bast inhibin" (business commodity name:Graciousness), is the biological preparations first kind of anti-tumor new medicine, it is in the world the multi-target blood vessel bast inhibin anti-cancer new medicine.The sarcoma new assistance chemotherapy plan union blood vessel growth chalone in the sarcoma blood vessel production, the attack and the shift present sarcoma treats a research hot spot, in order to observe the sarcoma new assistance chemotherapy plan union blood vessel growth inhibin to the osteosarcoma in VEGF, MMPs and the TIMP expression, conducts the animal model study, and provides as the clinical application the animal experimentation rationale.And treats in the plan at the osteosarcoma to use the chemotherapy medicine union suppression tumor blood vessel production medicine to take one kind of new treatment idea.Objective:1,the observation and damps the lump rate along the platinum union graciousness to the S180 mouse sarcoma animal model tumor growth curve;2,immunity group VEGF, MMPs and the TIMP expression and the tumor blood vessel production, invades between Run's relations;3,provides the zoology basic research for the anti-tumor medicine union graciousness line anti-blood vessel treatment sarcoma. Method:1,establish the S180 animal modelit is to put the S180 cell connecting pipe which is given by the Nanjing University Life Academy of science rapidly to invest together into 37℃the constant temperature water bath that liquid nitrogen frozen to save the S180 cell line connecting pipe, and to dissolve 1mim.Under the room temperature in 5mim restores to in the room temperature 20% embryo cow seroculture dilutes to original volume 5 times,450rpm low speed gentrifugalism lOmin, goes on clear, shifts joins the fresh 20% embryo cow seroculture to the culture bottle in, puts in 37℃,5%CO2 in the incubator.Recovers the cell vaccination in contains in the 10%FBS D-MEM culture medium, in 37℃,5%CO2 conventional raise transfer of generation.Trades the fluid every other day, the cell growth is rapid,3-4d passes on 1 generation, takes the logarithm vegetal period the cell to carry on the experiment.The S180 cell transfer of generation after the enough quantity, the S180 osteosarcoma cell which the collection raises, the PBS fluid washes 1 time, the adjustment cell density, makes 4x 10(?)7/mL the cell hangs the fluid/only pours into 36 C57/BL6 mouse back hypodermic injection according to 4×10(?)6 a cell, each mouse back hypodermic vaccinates 0.1ml.After vaccinates the S180 cell the 6th day C57/BL6 mouse back to form about heavy approximately 1g basically the entity lump.2,treated S180 mouse sarcoma animal model by drugsTo divides into stochastically to the S180 mouse sarcoma animal model medicine intervention 36 C57BL/6 mouse model 4 groups, respectively is:Control group, graciousness group, along platinum group, along platinum+graciousness union group.Each group of 9, each group is vaccinating 7 day to start to apply drugs starts for the medicine, the injection spot in the foreleg armpit hypodermic injection, is as follows for the medicine medicine dosage:Control group:Physiological saline inoculation fluid 1Omg/Kg·d; Graciousness group:Graciousness 10mg/Kg·d; Along platinum group:Along platinum 5mg/Kg·d; Along platinum+graciousness union group:Graciousness lOmg/Kg·d, along platinum 5mg/Kg·d, is same for the medicine time way.According to 1st,4,7,10,13,16,19, on 22nd altogether 8 times in the foreleg armpit hypodermic injection medicine, the experiment operates follows the asepsis principle.The experimental period mouse free feed, after end time 24h executes each group of animals for the medicine, splits takes the tumor specimen.3,take the tumor specimen to carry on the immunity groupThe principle enzyme mark immune body and the antigen form an antigen/antienzyme compound, again uses biological element immune body crossing linking, in which biology element mark rabbit anti-mouse IgM is the mark has HRP (horse radish peroxide enzyme) and the anti-rabbit and the anti-mouse immune globulin concrete member, is equal in two anti-, two anti-unifies after the biological element one anti-, combines to form the antigen-immune body-biology element two anti-compounds, after the union macro-molecule is the mark partner horse radish peroxide enzyme, again with DAB (3,3,-two aminobenzidines) responded results in the coloration.4 observation targets:4.1 survey transplant lump volume computation treatment damps the lump rate to be self-sufficientthe medicine to get up 1,4,7,10,13,16,19,22 days surveys the mouse body weight and the tumor long minor axis separately, and according to the formula is: V=1/2 ab2 (a is major axis, b is minor axis), tests the 28th day execution experiment mouse, the peeling tumor organizes and surveys the tumor organization major axis and the minor axis, the computation damps the lump rate (tumorinhibition rate, TIR), TIR= (control group tumor volume-experimental group tumor volume)/the control group tumor volume×100%, the plan moves the computation tumor volume, obtains each group the tumor growth curve.4.2 immunity group masculine result judgment standard:The slice carries on which according to the immunity group working instruction, under the microscope is located the tumor cell and the blood vessel endothelial cell with the VEGF masculine expression, in the cytoblastema appears yellow dyes the pellet; MMP-9 and the TIMP masculine gender dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, the dyeing intensity grading result uses the semi-quantitative analysis method to carry on H-to grade [2]: Selects 5 high power mirror fields of vision to each slice (×400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, according to the masculine cell percentage and the colored depth graduation, for details sees table 1.Above two grading adding together, no matter dyeing intensity, so long as the masculine cell< 10% all is a negative, by 0 represents the negative dyeing degree cell percentage; 3 divides into the weak masculine gender, by 1 represents weakness dyeing degree cell percentage masculine gender (+); 4-5 divides into the moderate masculine gender, by 2 represents the moderate dyeing degree cell percentage masculine gender (++); 6-7 divides into the strong masculine gender, by 3 represents the moderate dyeing degree cell percentage masculine gender (+++), by 0,1,2,3 on behalf of the negative, the weak dyeing, the medium dyeing and strong dyes 4 ranks.4.3 immunity histochemistry examination transplant lump organizes the VEGF expression organization specimen by 10% formaldehyde solution fixed 24h, the conventional paraffin wax embedding, prepares 5 m thick slices, carries on according to the immunity group working instruction. With the known masculine specimen took the masculine comparison, replaces by PBS anti-makes the negative comparison.The VEGF masculine expression is located the tumor cell and the blood vessel endothelial cell, in the cytoblastema appears yellow dyes the pellet; Selects 5 high power mirror fields of vision to each slice (×400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, respectively by 0,1,2,3 represents the negative, the weak dyeing, the medium dyeing and the strong dyeing and so on 4 ranks, H minute value= [(weak dyeing intensity cell percentage×1)+(moderate dyeing degree cell percentage×2)+(strong dyeing intensity cell percentage×3)] x 100, takes two viewer's mean values to take again finally the H-grading, H is worth the minute being situated between 0-300Between.4.4 immunity histochemistry examination transplant lump organizes MMP-9 to express MMP-9 and the TIMP masculine gender dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, finally uses the semi-quantitative analysis method to carry on the H-grading:Selects 5 high power mirror fields of vision to each slice (x400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, respectively by 0,1, 2,3 represents the negative, the weak dyeing, the medium dyeing and the strong dyeing and so on 4 ranks, H minute value= [(weak dyeing intensity cell percentage x1)+(moderate dyeing degree cell percentage x2)+(strong dyeing intensity cell percentage×3)] x 100, takes two viewer's mean values to take again finally the H-grading, H is worth the minute being situated between 0-300Between.The observation tests in each group of sarcoma animal model VEGF, MMPs and the TIMP immunity expression.4.5 immunity histochemistry examination transplant lump organizes TIMP to express TIMP and MMP-9 is same, the masculine dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, the result and the MMP-9 similar semi-quantitative analysis method carries on the H-grading.Statistics analysisThe obtained data uses the SPSS13.0 software to carry on statistics analysis.The transplant lump volume quota result (X±s) indicated with the mean value±standard deviation that, the group compares uses the single factor the variance analysis, during 22 groups compares uses the LSD examination. each group of transplant lump VEGF, MMP-9 and the TIMP H value semi-quantitative material uses the median (minimum value, maximum value) to indicate that, the group compares uses the Kruskall-Wallis examination, after during 22 groups compares uses Mann-Whitney the U-test (examination standard to use Bonferroni law adjustment alpha =0.008).By P< 0.05 thought has statistics significanceThe result:1. transplant lump growth situation and damps the lump rateThe lump tumor growth incubation period is 3-5d, the tumor assumes the expansibility to grow, the quality of material is slightly strong.The control group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:395.70±34.92 mm3,542.02±52.74 mm3,637.48±51.69 mm3,713.88±61.78 mm3,795.39±68.88 mm3,864.14±77.63 mm3,932.24±83.27mm3,990.37±89.25mm3; Graciousness group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally: 387.54±24.89mm3,506.70±28.07 mm3,605.55±40.89 mm3,685.8±40.89 mm3, 764.48±43.98 mm3,832.41±45.89 mm3,887.21±50.87 mm3,930.78±52.96 mm3; Along the platinum group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:365.90±23.72 mm3,478.70±27.01 mm3,553.73±34.65 mm3, 580.60±40.66 mm3,551.95±37.81mm3,497.19±43.47 mm3,431.43±38.81 mm3, 370.54±35.46mm3; The union group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:392.78±42.81 mm3,477.82±47.74 mm3,524.79±38.14 mm3,534.89±22.05 mm3,476.39±28.73mm3,392.15±28.73 mm3, 328.17±31.04mm3,275.83±32.50mm3.After the medication the 4th day control group tumor still rapidly grows, graciousness group compares the control group to be slightly slow, still long-enduring growth.4th days the tumor starts slow-growing after the platinum group and the union group medication, after 7th days relatively static, after but reduces gradually, but and lengthens along with the time drops rapidly, increases rapidly with the control group and graciousness group forms distinctively shows (Figure to for instance the chart 3).The 22nd day union group tumor reduces quickly, (P 0.05) has statistics significance along the platinum group and the union< group in the tumor volume.2. immunity group examination2.1 VRGF immunity group H grades.VRGF immunity group like charts show (Figure 6-chart 9), the control group, graciousness group's expression intensity is+++,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor VRGF the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Along platinum group and union group (P< 0.008); May infer union group VRGF the expression to be least, next is along the platinum group, is graciousness group once more, but the blank group are most.12 MMP-9 immunity group H grades.MMP-9 the immunity group like chart to show (Figure 10-chart 13), the control group, graciousness group's expression intensity is+++,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor VRGF the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group with has along the platinum group has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); May infer union group MMP-9 the expression to be least, next is along the platinum group, is graciousness group once more, but the blank group are most。2..3TIMP immunity group H grading.The TIMP immunity group like chart shows (Figure 14-chart 17), control group, graciousness group's expression intensity for+,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+++.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor TIMP the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); May infer blank group TIMP the expression to be least, next is graciousness group, but is once more along the platinum group, the union group most。Conclusion:1,the anti-tumor medicine union graciousness S180 sarcoma suppression blood vessel production function is obvious, obviously suppresses the tumor volume to increase, and damps the cancer rate to be high.2,in tumor VEGF and MMPs expression basic parallel, both participate in the tumor blood vessel together the production and coordinates the expression, anti-tumor medicine union graciousness causes in the lump VEGF and MMPs obviously reduces.3,in the tumor the MMPs/TIMP ratio reduces may suppress the tumor growth and invade Run, graciousness has the inhibitory action through many kinds of ways to VEGF and MMPs, and increases TIMP the expression, reduces the tumor blood vessel the production.4th, on clinical applies for the anti-tumor medicine union graciousness treatment sarcoma lays the experimental foundation, and treats the osteosarcoma animal model for the anti-tumor medicine union graciousness line anti-blood vessel, and the osteosarcoma clinical treatment plan opens the new treatment idea.
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