CN103006625A - Application of mannitol in preparation of anti- pancreatic cancer drug - Google Patents
Application of mannitol in preparation of anti- pancreatic cancer drug Download PDFInfo
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- CN103006625A CN103006625A CN2012105905928A CN201210590592A CN103006625A CN 103006625 A CN103006625 A CN 103006625A CN 2012105905928 A CN2012105905928 A CN 2012105905928A CN 201210590592 A CN201210590592 A CN 201210590592A CN 103006625 A CN103006625 A CN 103006625A
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Abstract
The invention discloses an application of mannitol in preparation of an anti-pancreatic cancer drug, and in particular relates to a use of a mannitol injection with a specific concentration of 10%-19% for treating pancreatic cancer. Traditionally, mannitol is only used as a diuretic, a high-permeability hypotensor and the like, while the inventor discovers through scientific experiments that mannitol has anti-tumor activity, and through several cell apoptosis detection technologies and by detecting apoptosis-related genes bcl-1, and bax, proves that the specific-concentration mannitol can induce various human cancer cell strains to generate obvious cancer cell apoptosis, grasps the effect relation with concentration and time, and first illustrates the possible mechanism; furthermore, the inventor has used the research result for tumor patient treatment and has achieved obvious curative effect, which provides theoretical basis and practical cases for the application of mannitol in preparation of the anti-tumor drug and the clinical generalization thereof.
Description
Technical field
The present invention relates to new purposes, the especially mannitol application in the anti-cancer of pancreas medicine of preparation of mannitol.
Background technology
At present, in antitumor research, the apoptosis of inducing cancer cell is an important approach, promoting apoptosis of tumor cells can reach tumor dwindles, the purpose that cancer disappears such as the equal energy such as traditional antitumor drug cisplatin, American and French human relations irritation cell mitochondrial apoptotic pathway, causes apoptosis.This kind mode has obvious superiority than the therapeutic modality that adopts killing tumor cell, if because antitumor drug can be a large amount of the apoptosis of induced tumor cell, activate the programmed death process of himself, will avoid the various side effect that cause because of material in a large amount of necrocytosis release cells, alleviate chemotherapy to Normocellular damage, prolong patient's life cycle.Yet, not yet filter out so far can the induced tumor cell antineoplastic agent of a large amount of apoptosis.The herb induction apoptosis of tumor cells mostly is experiment in vitro research, and the interior experiment of body is less, basically is in the primary research stage.Studies show that the proto-oncogene relevant with cell proliferation and antioncogene all participate in apoptotic regulation and control, wherein study more have Bcl-2 family, cytochrome C, antioncogene p53, Ice protease and Fas/APO-1 etc.Therefore, take above-mentioned apoptotic controlling gene as target gene, the medicine of seeking to have distinguish between tumor cells and normal cell in the trigger cell apoptotic effect (can triggering tumor cell apoptosis and reactionless to normal cell) ability is in recent years the various countries scientist direction of making great efforts and the new highlight of antitumor drug research.
In traditional non-cancer therapy drug, scientist finds that wherein having some to have can cause apoptotic effect, and this may become the approach of the new antitumor drug of research and development.Yet that has found has a non-cancer therapy drug that causes cells apoptosis, only is confirmed in laboratory, and still can not brings out the apoptosis of massive tumor cell, not yet really is used for the treatment of clinically tumor.
Mannitol (Mannitol) is a kind of hexanhexol, and traditional medicine also is the medicine of Lowering the intracranial hypertension, intraocular pressure and treatment nephropathy as good diuretic.The common dosage forms formula mannitol injection liquid permeates depressor as height, be clinical rescue particularly brain disorder rescue a kind of medicine commonly used, have that the desired blood pressure lowering of Lowering the intracranial hypertension medicine is fast, curative effect characteristics accurately.Have no the report whether mannitol has the cancer cell specific induction of apoptosis effect both at home and abroad, also mannitol is not used for the clinical practice precedent of antineoplaston.
Summary of the invention
The technical problem to be solved in the present invention provides the application of a kind of mannitol in the anti-cancer of pancreas medicine of preparation, particularly uses the formula mannitol injection liquid treatment cancer of pancreas of certain concentration.
Adopt following technical scheme for solving the problems of the technologies described above the present invention:
The application of mannitol in the preparation antitumor drug.
Above-mentioned tumor is pulmonary carcinoma, hepatocarcinoma, breast carcinoma, cancer of pancreas, gastric cancer, adenocarcinoma of stomach.
Pulmonary carcinoma is lung squamous cancer, adenocarcinoma of lung.
Above-mentioned antitumor drug is formula mannitol injection liquid.
The mass concentration of above-mentioned formula mannitol injection liquid is 10%～19%.
The mass concentration of above-mentioned formula mannitol injection liquid is 13%～17%.
The inventor finds that by scientific experiments mannitol has anti-tumor activity, and especially the formula mannitol injection liquid of certain concentration has and obviously causes apoptotic effect.By adopting multinomial apoptosis detection technique and detecting apoptosis-related genes bcl-2, bax, confirmation certain concentration mannitol can be induced the obvious cancer cell-apoptosis of the multiple JEG-3 of people, and itself and concentration, the effect relation of time have been grasped, tentatively illustrate its possibility mechanism, and, the inventor has been used for result of study the tumour patient treatment and has obtained obvious curative effect, and these provide theoretical foundation and actual case for application and the clinical expansion thereof of mannitol in the preparation antitumor drug.Simultaneously, the present invention is that the medicine that science is sought the inducing apoptosis of tumour cell of high-efficiency low-toxicity has at an easy rate been opened up new thinking, has significance of scientific research.
Description of drawings
Fig. 1 is 48 hours MTT concentration-suppression ratio curve charts.
Meta-suppression ratio curve chart when Fig. 2 is 7 days MTT is among the figure: 120ug/ul, 240ug/ul, 360ug/ul, 480ug/ul, 5100ug/ul, 6120ug/ul.
Fig. 3 is 7 days MTT concentration-suppression ratio curve charts, among the figure: 1 one days, 2 two days, 3 three days, 4 four days, 5 five days, 6 six days, 7 seven days.
Meta-suppression ratio curve chart when Fig. 4 is chemiluminescence in 7 days.
Fig. 5 is the matched group transmission electron microscope picture.
Fig. 6 is the matched group transmission electron microscope picture.
Fig. 7 is 24 hours group transmission electron microscope pictures.
Fig. 8 is 24 hours group transmission electron microscope pictures.
Fig. 9 is 48 hours group transmission electron microscope pictures.
Figure 10 is 48 hours group transmission electron microscope pictures.
Figure 11 is that H1299 apoptosis flow cytometer testing result contrasts 1 picture group.
Figure 12 is that H1299 apoptosis flow cytometer testing result contrasts 2 picture groups.
Figure 13 is that H1299 apoptosis flow cytometer testing result contrasts 3 picture groups.
Figure 14 is H1299 apoptosis flow cytometer testing result 0% picture group.
Figure 15 is H1299 apoptosis flow cytometer testing result 5% picture group.
Figure 16 is H1299 apoptosis flow cytometer testing result 9% picture group.
Figure 17 is H1299 apoptosis flow cytometer testing result 13% picture group.
Figure 18 is H1299 apoptosis flow cytometer testing result 17% picture group.
Figure 19 is H1299 apoptosis flow cytometer testing result 20% picture group.
Figure 20 is early stage+non-viable apoptotic cell and dose relationship figure.
Figure 21 is necrosis and apoptosis cell and dose relationship figure in late period.
The specific embodiment
1 experiment material
1.1 material
1.1.1 cell strain: the SPC-A-1 human lung cancer cell line draws from the institute of oncology, Shanghai.
1.1.2 reagent: RPMI1640, trypsin are available from Gibcol BRL company; New-born calf serum is available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; Sodium bicarbonate (analytical pure) is that Hebei Ci Zhou pharmaceutical factory produces; Penicillin sodium and streptomycin sulfate are HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory production; Tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) are Sigma company product; ATP test kit (ATP extracting solution, fluorescein-luciferase, diluent etc.) is German DCS company product; Sodium citrate is Guangzhou Chemical Reagent Factory production; Propidium iodide (PI), RnaseA are Sigma company product; Formula mannitol injection liquid is available from Pharmaceutical Group Co.,Ltd of Nanning Baihui.
1.1.3 equipment: CO
2Incubator (Japanese HERAEUS); Inverted microscope (Japanese NIKON); Full-automatic microplate reader (Finland DENLEY DRAGON MK-2 type); The board-like analyser of self-luminous (German BETHOLD); Flow cytometer (U.S. BECKMAN); Transmission electron microscope.
2 experimental techniques and observation index
2.1 cell culture: 5%CO
2Incubator, with containing 10% new-born calf serum, penicillin, streptomycin each 1 * 10
5U/L and NaHCO
3Transfer pH value to the RPMI1640 culture medium culturing of 7.2-7.4, collect the exponential phase cell and test.
Measure cell inhibitory rate 2.2 adopt mtt assay: be 2 * 10 with concentration
4The cell suspension inoculation of/ml in 96 porocyte culture plates, every pore volume 100 μ L.Behind the cell attachment, add respectively the mannitol of 1-150 μ g/ μ l concentration, not dosing of matched group only adds the culture medium of respective volume, establishes simultaneously multiple hole, 37 ℃, 5%CO
2Cultivate after 48 hours under the condition, every hole adds 20 μ l MTT, continue to cultivate 4 hours, add 100 μ l DMSO, the vibration mixing, with full-automatic microplate reader colorimetric, 492nm wavelength place measures the absorbance A value in each hole, calculating mean value, and press following formula and calculate inhibitory rate of cell growth: inhibitory rate of cell growth (%)=(control group A-experimental group A)/control group A * 100%; Get the mannitol of 20,40,60,80,100,120 μ g/ μ l concentration with method, not dosing of matched group only adds the culture medium of respective volume, establishes simultaneously multiple hole, cultivates respectively 7 days, calculates the suppression ratio of variable concentrations every day.
2.3 adopt the chemiluminescence determination cell inhibitory rate: be 2 * 10 with concentration
4The cell suspension inoculation of/ml in 96 porocyte culture plates, every pore volume 100 μ l.Behind the cell attachment, add half-inhibition concentration (IC among 48 hours MTT results
50) mannitol, not dosing of matched group only adds the culture medium of respective volume, establishes simultaneously multiple hole, 37 ℃, 5%CO
2Cultivated under the condition 1,2,3,4,5,6,7 day, every hole adds 50 μ lATP extracting solution, abundant mixing, sucking-off 100 μ l mixed liquors in every hole add in another corresponding hole of 96 orifice plate, add 20 μ l fluorescein-luciferases in every hole of new plate, the vibration mixing detects the value in every hole with the board-like analyser of self-luminous, then calculating mean value, and calculate inhibitory rate of cell growth by following formula: inhibitory rate of cell growth (%)=(matched group-experimental group)/(matched group-blank organized) * 100%.
2.4 flow cytometry analysis cell cycle
2.4.1PI the preparation of dye liquor: accurately take by weighing PI5mg, RnaseA10mg, sodium citrate 0.1g on the high accuracy balance, be dissolved in the 100ml sterile saline.Accurately draw in the NP-400.3ml adding in the liquid, shake up, keep in Dark Place under 4 ℃.2.4.2 flow cytometry analysis cell cycle method: collect cultured cell, in PBS, wash twice, then add 70% ice ethanol and mixing by (1-2) * 106/ml, under 4 ℃, fixedly spend the night.During detection specimen with 1000r/min from 5min, the resuspended 5min of 3mlPBS, with 300 eye mesh screen filtration cell suspensions to remove the agglomerating cell mass of adhesion, centrifugal 5min under 1000r/min again, abandoning supernatant adds PI dye liquor 1ml/ pipe, 4 ℃ of lower lucifuge 30min.Get 48 hours half-inhibition concentration (IC among the MTT result
50), be divided into 24,48 hours two groups and matched group, totally three groups, carry out cell cycle analysis with flow cytometer, collect data, carry out the analysis of cell cycle at the MultiCycle analysis software, calculate respectively G
1, S, G
2The relative scale of/M phase.
2.5 transmission electron microscope detects: collect cultured cell, move into behind a small amount of PBS suspendible in the plastics taper centrifuge tube, at the centrifugal 5-10min of 1000-1500r/min, abandoning supernatant, adding new-born calf serum 1-2 drips, then dispel centrifugal thing with suction pipe and make its mixing, behind the centrifugal 3-5min, inhale as far as possible and abandon the unnecessary new-born calf serum in upper strata; Slowly add the glutaraldehyde fixative along tube wall, pre-fix 30min under 4 ℃,, make fixative fully infiltrate the bottom, and continued fixing at least 2 hours along tube wall standardized circle gently around specimen with fine needle, get 48 hours half-inhibition concentration (IC among the MTT result
50), be divided into 24,48 hours two groups and matched group, totally three groups, Electronic Speculum embedding step process routinely.
2.6 statistical procedures: all the data SPSS10.0 softwares carry out statistical disposition, the relatively employing t check of measurement data, the relatively employing χ of enumeration data
2Check.
3 experimental results
3.1 adopting mtt assay to measure the cell inhibitory rate result shows: in 48 hours, mannitol can suppress the proliferation activity of lung carcinoma cell when 1-150 μ g/ μ l concentration range, and its inhibitory action increases and strengthens with concentration, in 7 days, along with the growth of concentration and time, suppression ratio also constantly increases.See Fig. 1,2,3.
3.2 adopt chemiluminescence determination cell inhibitory rate result to show: to get 48 hours half-inhibition concentration (IC among the MTT result
50) time, act on 7 days, mannitol can suppress the proliferation activity of lung carcinoma cell, and inhibitory action in time prolongation and strengthen, see Fig. 4.
3.3 flow cytometry analysis cell cycle result shows: get 48 hours half-inhibition concentration (IC among the MTT result
50) time, act on 24,48,72 hours group cell cycles and stop at G
2+ M the phase than the matched group showed increased, see Table 1.
The impact of table 1 mannitol different disposal time cell cycle
3.4 the transmission electron microscope testing result shows: get 48 hours half-inhibition concentration (IC among the MTT result
50) time, act on 24 hours and organize visible cell film, intact nuclear membrane under the Electronic Speculum, kernel is clearer, drip than greasiness in the kytoplasm, visible apoptotic body act on 48 hours and organizes under the Electronic Speculum visible cell apoptosis more obvious, a large amount of apoptotic bodies, and as seen meeting the characteristics of tumor cell under the cellular control unit Electronic Speculum, nuclear is large, sees Fig. 5 to Figure 10.Fig. 5,6 is for matched group visible cell nuclear is large, and caryoplasm is out of proportion, and kernel is clearer, drips than greasiness in the kytoplasm; Fig. 7,8 is 24 hours group visible cell intact nuclear membrane, and visible apoptotic body Fig. 9,10 is 48 hours visible a large amount of apoptotic bodies of group.
1 materials and methods
1.1 main agents and instrument: DMEM (high sugar) culture fluid (Sai Mo of U.S. hyclone company flies Beijing company limited packing of generation that biochemistry goods), trypsin, newborn calf serum (FBS is available from the biological company limited of Hangzhou Ilex purpurea Hassk.[I.chinensis Sims), 20% mannitol (available from peace Double-Crane Pharmaceutical Co., Ltd company limited), Giemsa staining kit (being provided by No.1 Hospital Attached to Guangxi Medical Univ. inspection center), apoptosis hoechst33342 test kit (available from the green skies, Shanghai Bioisystech Co., Ltd) apoptosis Dapi staining kit, flow cytometer test kit (all available from Nanjing KaiJi Biology Science Development Co., Ltd), Bcl-2 detection kit (stepping neoplasm development in science and technology company limited available from Foochow), pentanediol, transmission electron microscope is provided by Guangxi Medical University's Electron Microscopy Room.CK41 type inverted microscope (Japanese Olympus), photographic system (Japanese Nikon72000U inverted microscope and DS-5MC high resolution CCD digital photographing 5,000,000 picture numbers), provided by National Key Laboratory of Guangxi University, flow cytometer (U.S. company BD FACSCalibur) is provided and is assisted by experimental center, the autonomous the People's Hospital of Zhuang nationality in Guangxi and detects.
1.2 cell culture: the H1299 cell strain is human lung adenocarcinoma cell, is so kind as to give by Guangxi treatment and prevention of tumour institute doctor Pan Hong.This cell is spindle shape, leaf or polygon, adherent growth, and Cytoplasm is full, and growth is unfolded, and the flanking cell growth is merged in flakes.Get and be diluted to 2 * 10
4/ ml is in the cell suspension of exponential phase, is incubated at 37 ℃ and contains 5%CO
2In the cell culture incubator, culture fluid is DMEM (high sugar) culture fluid that contains 10% newborn calf serum, 100u/ml penicillin and 100u/ml streptomycin, and 0.25% trypsinization goes down to posterity, the growth conditions of observation of cell under the CK41 type inverted microscope.
1.3 method:
(1) Giemsa dyeing observation of cell apoptosis form: well-grown state cell put into to take in advance be in well-grown H1299 cell suspending liquid, add six orifice plates that are placed with in advance coverslip, being put into 37 ℃ of incubators cultivates, allow cell climbing sheet grow, treat that it is long to 85%～90%, the mannitol (0% that adds respectively variable concentrations, 5%, 9%, 13%, 17%, 20%), cultivated 48 hours, and took out coverslip, PBS washes 2 times, methanol is 5min fixedly, the Giemsa 15min that dyes dries rear resinene mounting, the lower apoptotic characteristic variations of taking pictures of observing of microscope (several 400 times of Japanese Nikon72000U inverted microscope and DS-5MC high resolution CCD digital photographing 5,000,000 pictures).
(2) apoptosis Dapi dyeing observation of cell nuclear apoptotic body: get be in well-grown H1299 cell do not add contain variable concentrations 0%, 5%, 9%, 13%, 17%, 20% mannitol-: DMEM (high sugar) culture fluid (containing 10%FBS) 10ml.Continue to cultivate 48 hours PBS washing 2 times, 0.25% trypsinization 3min, add immediately DMEM (high sugar) culture fluid (10%FBS) 10ml and stop digestion, centrifugal, abandon supernatant, PBS washing 2 times, centrifugal again, abandon supernatant, make cell suspension (100,000 cells/1ml) with PBS dilution afterwards, cell suspension 500ul put into practice shooting fixedly centrifuge tube centrifugal (8000r/min, 5min) to microscope slide, drying, dehydrated alcohol is fixed, and is dry again.Then under the lucifuge condition, press the dyeing of apoptosis Dapi colouration box description, PBS washing by soaking 3 times in glass jar, each 15min, dry again, dry rear neutral number rubber seal sheet, fluorescence microscope is with 400 times of 340/380 ultraviolet excitation and DS-5MC high resolution CCD digital photographings) under the nucleus apoptotic body situation of observing, take pictures.4 ℃ of apoptosis Dapi dyeing glass slides keep in Dark Place.
The result judges: the chromatinic morphological change of nucleus was divided into for three phases in the apoptosis process: the nucleus of I phase is corrugated (rippled) or is crease sample (creased), and enrichment stage appears in the part chromatin; The nuclear chromatin height coagulation of II a phase, marginalisation; The nucleus of II b phase is cracked into fragment, produces apoptotic body.
(3) apoptosis hoechst33342 reagent dyeing observation of cell nuclear apoptotic body: step is identical with (2), then under the lucifuge condition, press the dyeing of apoptosis hoechst33342 test kit description, PBS washing by soaking 3 times in glass jar, each 15min, dry again, dry rear neutral number rubber seal sheet, fluorescence microscope is with 400 times of 340/380 ultraviolet excitation and DS-5MC high resolution CCD digital photographings) under the nucleus apoptotic body situation of observing, take pictures.4 ℃ of apoptosis hoechst33342 reagent dyeing slides keep in Dark Place.
(4) transmission electron microscope observation: step is identical with (2), and is centrifugal, abandons supernatant, and PBS washing 2 times moves into the 1.5EP pipe centrifugal again, abandons supernatant, with observing apoptosis cellular morphology under the fixing rear transmission electron microscope of pentanediol.
The result judges: the apoptotic cell smaller volume, Cytoplasm is concentrated.Many cavity structures that are called air pocket (cavitations) appear in the nuclear chromatin height coiling of apoptosis I phase (pro-apoptosis nuclei); The nuclear chromatin height coagulation of II a phase, marginalisation; In apoptotic late period, nucleus is cracked into fragment, produces apoptotic body.
(5) flow cytometer detects the apoptosis of cell: step is identical with (2), collects all suspension cells and pours into respectively and indicate 0%, 5%, 9%, 13%, 17%, 20% centrifuge tube (10ml), centrifugal 2000r/2min, abandoning supernatant, wash 2 times with PBS, centrifugal 2000r/2min abandons clear liquid again, resuspended, centrifugal 2000r/2min, resuspended again, cell counting (1X10
6)/ml presses table 1 and adds corresponding reagent, lucifuge, puts into ice bath FACSCalibur on 1h and carries out the flow cytometry detection by quantitative, the results are shown in Table 2.
Table 2 flow cytometer detects the apoptosis result of cell
(6) Immunohistochemistry bcl-2 Protein Detection: step is identical with (1), and 4 ℃ of acetone are fixed, and dry.The hyperbaric heating method is carried out antigen retrieval, 10min, and distilled water cooling, PBS washing 2-3 time is put into sheet and is hatched box, drips primary antibodie 50ul, and 4 ℃ in refrigerator spends the night, and takes out and washs 2-3 time with PBS, dries.Again sheet is put into and hatched box, drip two and resist
(the SuperpicTure numbering: 87-8963) 50ul, incubated at room 10min, PBS washing 2-3 time is dried.The DAB 2-10min that develops the color, haematoxylin is redyed, dehydration, transparent, mounting.
The result judges: pulmonary carcinoma H1299 Immunohistochemistry is assessed, brown yellow granule positive (+) is arranged in the cytoplasm, the cell non-coloring or with background indifference negative (-).
1.4 statistical analysis technique: adopt SPSS for windows17.0 to do curve regression analysis (Curve estimation), be the standard of judging optimum equation with the coefficient of determination to the maximum.Cell proportion and dose relationship adopt the analysis of Spearman rank correlation.
2. result
2.1 normal pulmonary carcinoma H1299 Growth of Cells developmental state of cultivating
2.2Giemsa dyeing observation of cell apoptosis form: microscopically is observed visible DMEM (high sugar) culture fluid (10%FBS) cellular control unit and is the shuttle type, Cytoplasm is full, nuclear is evenly placed in the middle, and through mannitol DMEM (high sugar) culture fluid (10%FBS) processed group apoptotic cell cell space dwindles, Cytoplasm red dye, examine concentrate, chromatin margination, the in addition cavitation phenomena that has.Contain 13%, the cell quantity of 17% mannitol-DMEM (high sugar) culture fluid (10%FBS) processed group chromatin margination obviously more than 5%, 9%, 20% mannitol-DMEM (high sugar) culture fluid (10%FBS) group, between prompting apoptotic cell quantity and the Dosages variable concentrations notable difference is arranged.
2.3Dapi dyeing observation of cell nuclear apoptotic body: at the visible DMEM of fluorescence microscopy Microscopic observation (high sugar) culture fluid (10%FBS) cellular control unit nuclear apoptotic body seldom, less than 20%, contain 13%, 17% mannitol-DMEM (high sugar) culture fluid (10%FBS) processed group nucleus apoptotic body is about 83%, the nucleus edge of apoptotic cell is irregular, the nuclei dyeing colour solid concentrates, painted heavier, and with karyopyknosis, and 5%, 9%, 20% mannitol-DMEM (high sugar) culture fluid (10%FBS) processed group nucleus apoptotic body is pointed out apoptotic cell below 50%, between nuclear apoptotic body and the Dosages variable concentrations notable difference is arranged.
2.4hoechst33342 reagent dyeing observation of cell nuclear apoptotic body: in the visible DMEM of fluorescence microscopy Microscopic observation (high sugar) culture fluid (10%FBS) cellular control unit intact nuclear membrane, karyomorphism is full, is the casual faint blue-fluorescence of even ni.And contain 13%, 17% mannitol-DMEM (high sugar) culture fluid (10%FBS) processed group observes typical apoptotic morphologic feature under fluorescence microscope; Nucleus volume dwindles, nuclear chromatin is assembled, can see dense fine and close graininess or the block fluorescence that strengthens of dying in the Cytoplasm, and can see apoptotic body about 90%, the cell number that 13%, 17% treatment with mannitol group fluorescence strengthens obviously more than 5%, 9%, 20% mannitol-DMEM (high sugar) culture fluid (10%FBS) processed group.Prompting, the dosage that the cell quantity that fluorescence strengthens uses experiment has certain dependency.More point out between apoptotic cell, nuclear apoptotic body and the Dosages variable concentrations notable difference is arranged.
2.5 transmission electron microscope observation apoptosis morphological observation: the apoptosis nucleorhexis, nuclear heteronuclear chromatin margination, nuclear membrane break, nuclear chromatin is partly overflowed accidental apoptotic body.Also see that early apoptosis appears in nucleus, the nuclear chromatin coagulation is bulk, broken disintegrate is arranged.Cavity appears in Cytoplasm.
2.6 flow cytometer detects the variation (seeing table 2-4, Figure 11 to Figure 21 for details) of the apoptosis rate of cell
Table 3 is early stage+and the non-viable apoptotic cell model gathers and estimates of parameters
Dependent variable: x1
Annotate: 1.Spearman coefficient of rank correlation: r
s=0.943, P=0.005(has positive correlation);
2. early stage+non-viable apoptotic cell and dosage have relation (wherein the effect of composite curve equation is best) Y=18.994 * 1.087
X
Table 4 necrosis and apoptosis in late period cell model gathers and estimates of parameters
Dependent variable: y
Annotate: 1.Spearman coefficient of rank correlation: r
s=0.943, P=0.005(has positive correlation)
2. late period, necrosis and apoptosis cell and dosage had relation (wherein the effect of quadratic equation is best)
Y=20.965－3.645X－0.362X
2
2.7 Immunohistochemistry bcl-2 protein expression situation: it is (+) or (++) that microscopically is observed visible 13%, 17% treatment with mannitol group bcl-2 protein expression positive, and 5%, 9%, 20% treatment with mannitol group bcl-2 protein expression is (-).Bcl-2 protein expression positive reaction material mainly is positioned at cytoplasm, and minority is positioned on the nuclear membrane, is brown yellow granule or lumps.
3 discuss
The Bcl-2 protein family is one group of vital apoptotic proteins in the cell, experimental results show that, overexpression Bcl-2 can not promote cell proliferation, but shows as on the contrary strongly to stop the apoptosis of being induced by Gamma-radiation, heat shock and Treated with Chemotherapeutic Drugs thing lamp.Gene knockout is tested and is shown, Bcl-2 plays vital effect in keeping cell dynamic equilibrium, because the Bcl-2 family protein can form large hole at mitochondrial outer membrane, discharges short antiapoptotic factors to Cytoplasm, activates caspase, cell death inducing.In addition, the Bcl-2 family protein can affect endoplasmic reticulum Ca
2+The ion emission levels, the osmotic pressure of change mitochondrial outer membrane, final cell death inducing.Therefore, the Bcl-2 protein families is the topmost regulatory factor that discharges at mitochondrion level control antiapoptotic factors, is apoptotic regulation and control person.
Method with reference to embodiment 2, with the formula mannitol injection liquid of variable concentrations at the external SPC-A-1 human lung carcinoma cell that acts on respectively, PLC (human liver cancer cell), HepG2 (hepatoma carcinoma cell), HepG2/2-15 (hepatoma carcinoma cell), Hep-3B (hepatoma carcinoma cell), SMMC-7721 (hepatoma carcinoma cell), hepatocarcinoma 9204 (hepatoma carcinoma cell), Bel-7402 (hepatoma carcinoma cell), Bel-7404 (hepatoma carcinoma cell), HuH7-HCV (HCV infection hepatoma carcinoma cell), HuH-7 (hepatoma carcinoma cell), CBRH-7919 (Hepar Mus cancerous cell), CRL-1548 (rat hepatoma cell), the MiaPaCa-2(pancreatic cancer cell), the BxPC-3(pancreatic cancer cell), the CFPAC(pancreatic cancer cell), A549 (lung adenocarcinoma cell), A549/DDP (A549 drug-resistant cell strain), Tul-1 (lung carcinoma cell), LLC euwis lung carcinoma cell, MGC-803 (stomach cancer cell), BGC-823 (Poorly differentiated gastric carcinoma cells), SGC-7901 (gastric adenocarcinoma cells), and utilize Giemsa to dye, apoptosis Dapi dyeing, the hoechst33342 reagent dyeing, transmission electron microscope, the method such as flow cytometer and Immunohistochemistry observation of cell apoptosis reaches bcl-2 protein expression in this process, studies show that mannitol can induce above-mentioned targeted cancerous cells to present apoptosis equally, the result is as follows:
1〉mannitol causes that apoptotic better quality concentration is 13%～17%;
2〉mannitol causes that the cells apoptosis time range is 24 hours～120 hours;
3〉mannitol cause comprise apoptotic period early apoptosis, mid-term apoptosis and late period apoptosis;
4〉mannitol causes apoptotic possibility mechanism: the Bcl-2 family protein can form large hole at mitochondrial outer membrane, discharges short antiapoptotic factors to Cytoplasm, thereby activates the caspase cell death inducing.
Adopt mtt assay to measure certain concentration mannitol to the cell inhibitory rate of breast cancer cell with reference to embodiment 1.The result shows: in 48 hours, mannitol can suppress the proliferation activity of breast cancer cell when 1～150 μ g/ μ l concentration range, and its inhibitory action increases and strengthens with concentration, and in 5 days, along with the growth of concentration and time, suppression ratio also constantly increases.
Adopt chemiluminescence determination certain concentration mannitol to the cell inhibitory rate of SGC-7901 (gastric adenocarcinoma cells) with reference to embodiment 1.The result shows: get 48 hours half-inhibition concentration (IC among the MTT result
50) time, act on 7 days, mannitol can suppress the proliferation activity of gastric adenocarcinoma cells, and inhibitory action in time prolongation and strengthen.
Yang patient's pathology is diagnosed as multiple transfer in diffuse type hepatocarcinoma and the lung, without surgical engine meeting, and also capable chemotherapy and radiation.Injection 17% formula mannitol injection liquid (10ml～15ml/ time/single tumor body, 3 days once, is used in conjunction 3 times) treatment in the percutaneous lung puncture part pulmonary metastases.4 weeks are by X-ray film check and measure size of tumor, and metastasis is by respectively by original 2cm in the lung
3, 4cm
3Narrow down to 1cm
3, 1.5cm
3, reach " partial rcsponse " of oncotherapy effect (PR), and without any side effects.(WHO is about treatment of solid tumors evaluation criterion PR: dwindle 50% and kept for 4 weeks; RECIST is about treatment of solid tumors evaluation criterion PR: dwindle 30% and kept for 4 weeks)
After other had 2 routine hepatocarcinoma and the interior multiple transfer patient of lung to adopt identical treatment, effect reached RECIST standard P R.
The 5 routine patients such as Lu are that pleura shifts hydrothorax and forms behind the breast cancer operation, and cancerous cell is seen in cytological examination of hydrothorax, through 19% formula mannitol injection liquid thoracic cavity perfusion therapy (500ml/ time, one day 2 times, be used in conjunction 3～5 days), cancerous cell disappears, hydrothorax is controlled February～May (reaching CR), and is without any side effects.
The 2 routine patients such as Ma are advanced pancreatic cancer, have pleura to shift hydrothorax and form, and cancerous cell is seen in cytological examination of hydrothorax, through 19% formula mannitol injection liquid thoracic cavity perfusion therapy (500ml/ time, one day 2 times, be used in conjunction 3～5 days), cancerous cell disappears, and is hydrothorax control February above (reaching CR), without any side effects.
Lee is that 2 years pleuras of adenocarcinoma of stomach postoperative shift hydrothorax and form, and cancerous cell is seen in cytological examination of hydrothorax, through 19% formula mannitol injection liquid thoracic cavity perfusion therapy (500ml/ time, one day 2 times, be used in conjunction 3～5 days), cancerous cell disappears, hydrothorax is controlled May (reaching CR), and is without any side effects.
Indigo plant certain wait 23 routine patient's pathology to be diagnosed as lung squamous cancer or adenocarcinoma of lung and cancerous hydrothorax formation (late period), injection 17% formula mannitol injection liquid (10ml～15ml/ time/single tumor body in percutaneous lung puncture tumor, 3 days are once, are used in conjunction 3 times), and carry out the thoracic cavity perfusion therapy (500ml/ time with 19% formula mannitol injection liquid, one day 2 times, be used in conjunction 3～5 days), cancer dwindles 30%～50% in the lung, and cancerous hydrothorax is controlled more than 2 months, indivedual case cancerous hydrothorax controls had no recurrence in 2 years, and are without any side effects.
Claims (4)
1. the application of mannitol in the anti-cancer of pancreas medicine of preparation.
2. application according to claim 1 is characterized in that: described medicine is formula mannitol injection liquid.
3. application according to claim 2 is characterized in that: the mass concentration of described formula mannitol injection liquid is 10%～19%.
4.. application according to claim 3 is characterized in that: the mass concentration of described formula mannitol injection liquid is 13%～17%.
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| CN201210590592.8A CN103006625B (en) | 2011-04-18 | 2011-04-18 | Application of mannitol in preparation of anti- pancreatic cancer drug |
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| CN 201110096567 Division CN102225059B (en) | 2011-04-18 | 2011-04-18 | Application of mannitol in preparing antitumor medicines |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115300491A (en) * | 2022-08-26 | 2022-11-08 | 广西医科大学附属肿瘤医院 | Application of mannitol in preparation of anti-lung squamous carcinoma cell NCI-H292 medicine |
| CN115337293A (en) * | 2022-08-26 | 2022-11-15 | 广西医科大学附属肿瘤医院 | Application of mannitol in preparation of lung squamous carcinoma cell NCI-H226 resistant medicine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028243A (en) * | 2007-04-12 | 2007-09-05 | 济南康泉医药科技有限公司 | Anti-cancer composition containing neoformative vascular inhibitor and Eps mycin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101028243A (en) * | 2007-04-12 | 2007-09-05 | 济南康泉医药科技有限公司 | Anti-cancer composition containing neoformative vascular inhibitor and Eps mycin |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115300491A (en) * | 2022-08-26 | 2022-11-08 | 广西医科大学附属肿瘤医院 | Application of mannitol in preparation of anti-lung squamous carcinoma cell NCI-H292 medicine |
| CN115337293A (en) * | 2022-08-26 | 2022-11-15 | 广西医科大学附属肿瘤医院 | Application of mannitol in preparation of lung squamous carcinoma cell NCI-H226 resistant medicine |
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