CN102397534B

CN102397534B - Use of insulin in preparation of drug for promoting jaw bone tissue healing

Info

Publication number: CN102397534B
Application number: CN 201010274095
Authority: CN
Inventors: 刘洪臣; 汪林; 王东胜
Original assignee: Chinese PLA General Hospital
Current assignee: Chinese PLA General Hospital
Priority date: 2010-09-07
Filing date: 2010-09-07
Publication date: 2013-10-23
Anticipated expiration: 2030-09-07
Also published as: CN102397534A

Abstract

The invention discloses a use of insulin in preparation of a drug for promoting jaw bone tissue healing. In exploration of an insulin medicinal use, animal jaw bone tissue is utilized as an experimental material, and specifically, an experiment comprises the following steps of through a cell biology experiment, detecting insulin effects of promoting external jaw bone tissue osteoblast propagation, and locally applying insulin to living animal jaw bone tissue for promotion of jaw bone tissue healing. The use provided by the invention is a novel medicinal use of insulin and improves insulin effects on jaw bone tissue healing. In the use, insulin directly acts on jaw bone tissue and thus side-effect caused by insulin long-term injection is avoided and jaw bone tissue healing of 2-type diabetic patients is greatly promoted. Through local drug application, the efficiency of normal human jaw bone tissue healing promoted by insulin is effectively improved.

Description

The purposes of insulin in the medicine of preparation promotion mandibular organization healing

Technical field

The present invention relates to a kind of pharmaceutical usage of insulin.Particularly, the present invention relates to insulin and can be used as the medicine that promotes the healing of animal mandibular organization.The present invention has promoted the osteoblastic propagation of jawbone at first with the cultured cell in vitro of insulin action in mandibular organization; Secondly, the present invention acts on mandibular organization with the insulin topical, and insulin has strengthened the ability of rat exodontia nest mandibular organization healing.

Background technology

Insulin forms A by 51 aminoacid, two peptide chains of B, the A chain contains 21 aminoacid, the B chain contains 30 aminoacid, article two, borrow two disulfide bond to connect between the peptide chain, a disulfide bond [Lu H is also arranged between the 6th and the 11st amino acids of A chain, Kraut D, Gerstenfeld L et al.Diabetes interferes with the bone formation by affecting the expression of transcription factors that regulate osteoblast differentiation.Endocrinology.2003,144 (1): 346-52.Yuksel, E., Weinfeld.A.B., Cleek.R.et al.Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres.Plast.Reconstr.Surg 2000,105 (5): 1712-1720.].The human insulin molecule amount is 5.7KD, and isoelectric point, IP is pH5.6.(pH2.5-3.5) is more stable at sour environment, and be easily destroyed in alkaline solution, can form the insulin crystals such as zinc, cobalt.Again since in its molecule acidic amino acid more, can with the combinations such as basic protein such as protamine, form the protamine zinc insulin that molecular weight is large, meltage is low.The injection of this kind preparation is subcutaneous or the muscle absorption is slower, and long action time is protamine zine insulin.Insulin from islet secretion enters liver through portal vein, and 40%-50% decomposes in liver, and all the other enter the body loop distribution in whole body.From the intravenous injection insulin, 90% disappears from blood in 20min, and the overwhelming majority is absorbed by tissue or by the liver deactivation.The physiological action of insulin is mainly the promotion anabolism, main target organ is liver, fatty tissue, skeletal muscle [Krakauer JC, McKenna MJ, Buderer NF, et al.Bone loss and bone turnover in diabetes.Diabetes.1995,44 (7): 775-782.Bouillon R, Bex M, Van Herck E et al.Influence of age, sex, and insulin on osteoblast function:osteoblast dysfunction in diabetes mellitus.Clin Endocrinol Metab.1995,80 (4): 1194-1202.Rosen CJ, Ackert-Bicknell CL, Adamo ML et al.Congenic mice with low serum IGF-I have increased body fat, reduced bone mineral density, and an altered osteoblast differentiation program.Bone.2004.35 (5): 1046-1058.].Insulin makes the glucose that absorbs after the feed change in a large number glycogen and stores, and suppresses gluconeogenesis.Aspect the protein metabolism adjusting, insulin mainly works to protein synthesis and storage, it is by promoting that with receptors bind amino acid transport enters cell, and act on ribosome, increasing ribonucleic acid and DNA (deoxyribonucleic acid) generates, thereby further increase protein synthesis [Devlin, H.J.Hoyland, J.F.Newall et al.Trabecular bone formation in the healing of the rodent molar tooth extraction socket.Bone Miner Res.1997,12 (12): 2061-2067.].Although insulin is the main medicine for the treatment of at present diabetes, its molecular weight is large, and the half-life is short, fat-soluble poor, be difficult for seeing through biomembrane, easily degraded by the gastrointestinal enzyme, so the most widely use approach of insulin is as main take injecting drug use up to now.But adopt the insulinize diabetes-alleviating to the adverse effect of oral cavity partial implantation body healing, alleviate the pathological change [Boney that bone regeneration around implant occurs because of diabetes, C.M., Moats-Staats, B.M., Stiles et al.Expression of insulin-like growth factor-1 (IGF-1) and IGF-binding proteins during adipogenesis.Endocrinology.1994,135:1863-1868.Bouxsein ML, Rosen CJ, Turner CH et al.Generation of a new congenic mouse strain to test the relationships among serum insulin-like growth factor I, bone mineral density, and skeletal morphology in vivo.Bone Miner Res.2002,17 (4): 570-579.], strengthen the key that the Healing around Titanium Implants ability becomes diabetics tooth plantation treatment.

Diabetes are that a kind of common endocrine metabolism is sick, are that blood glucose due to or the relative deficiency absolute take insulin and glucose in urine increase as principal character and cause the chronic Developmental and Metabolic Disorder syndrome of whole body of sugar, protein and lipodystrophy.At present the diabetes mellitus in China patient has reached 5,000 ten thousand, accounts for 1/5 of world's diabetic population sum, and prevalence occupies the second in the world India after, and with every days at least three thousand people speed increase annual increasing above 1,200,000.Type 2 diabetes mellitus (type 2 diabetes mellitus, T2DM) is the main Types of diabetes, accounts for more than 90% of all diabeticss.Along with expanding economy, social senilization, the T2DM prevalence increases year by year.From world wide, T2DM has become one of chronic disease of serious threat human health.Diabetology branch of Chinese Medical Association shows that in the Epidemiological study result of the up-to-date diabetes that 14 provinces and cities in the whole nation carry out in year May in June, 2007 to 2008 prevalence of cities and towns diabetes has reached 11.28%.

What bone was integrated is the basis of dentistry implant success, yet hyperglycemia can suppress osteoblastic differentiation, can also produce illeffects to bone matrix and composition thereof, the adhesion, growth, the gathering that affect simultaneously extracellular matrix cause the diabetics bone regeneration around implant bone formation obstacle to occur.Control blood glucose the most effective the most frequently used medicine is exactly insulin, and its physiological action is mainly the promotion anabolism, and the degree of insulin and Cell binding depends on receptor number and its affinity on the cell.The number of receptor is constant under normal physiological conditions, but because the impact that cell physiological state different (such as the speed of growth, differentiation degree, cell cycle etc.) and external environment change also certain change can occur.Insulin, type-1 insulin like growth factor (IGF-1), IMA-IGF2BP3-001 (IGF-2) and corresponding receptor IR thereof, IGF-1R and IMA-IGF2BP3-001 receptor (IGF-2R) all are to belong to the IGFs system.Studies show that this system may have extremely important effect to growth, cell proliferation and the conversion etc. of embryo, nerve, skeletal muscle and skeleton.

Mandibular organization is facial important osseous tissue.Mandibular organization and to integrate be one of basis of dentistry implant success.For this reason, seeking a kind of more simple and easy to do medicine promotion and osseous tissue healing has great importance.At present, insulin is organized for odontotheca and is reported with a little, but the research of the direct administration of insulin mandibular organization does not also have.The present invention looks for another way, and insulin is topical mandibular organization only, has found that insulin can effectively promote mandibular organization's osteoblast healing.This has opened up a new way for the application of insulin.Insulin action may produce insulin resistant in diabetic, and local application can avoid the effect of insulin resistant in mandibular organization, thereby promotes type 2 diabetes mellitus patient dentistry implant success rate.In addition and since insulin can the body loop distribution in whole body, thereby promote anabolism; The present invention adopts topical, has reduced insulin administration and has given the counter productive that the normal person brings, and has improved insulin in the efficient that promotes mandibular organization's osteoblastic proliferation.

Summary of the invention

The present invention at first provides a kind of pharmaceutical usage of insulin.

The purposes of insulin of the present invention in the medicine of preparation promotion mandibular organization osteoblastic proliferation, the healing of promotion mandibular organization comprises that detecting insulin with Cell Biology Experiment promotes the osteoblastic propagation of external mandibular organization; Insulin acts locally on the mandibular organization of living animal, promotes the healing of mandibular organization.

The pharmaceutical usage of the insulin of indication of the present invention for be animal mandibular organization.

Animal of the present invention mandibular organization refers to that mandibular organization is at the osteoblast of In vitro culture and in the mandibular organization of living animal.

The present invention has improved the effectiveness of insulin.The present invention adopts insulin to directly act on mandibular organization, rather than by other approach such as intravenous injections.At first, the present invention has avoided the long term injections of insulin and the side effect that brings, and has brought very large facilitation for mandibular organization's healing of type 2 diabetes mellitus patient.Secondly, the present invention adopts topical, effectively raises the efficient of insulin in mandibular organization's agglutination.

Description of drawings

Each concentration insulin group of Fig. 1 is in the effect (mtt assay) of different time to osteoblastic proliferation, and * compares P＜0.05 with matched group.Wherein LA is the L-DMEM of insulin-containing (containing the 5.5mM glucose) not, and LB is for containing 10 ^-5The L-DMEM of M insulin; LC is for containing 10 ^-6The L-DMEM of M insulin; LD is for containing 10 ^-7The L-DMEM of M insulin.

Each concentration insulin group of Fig. 2 is compared P＜0.05 at different time to effect (mtt assay) * of osteoblastic proliferation with matched group.Wherein HA is the H-DMEM of insulin-containing (containing the 16.5mM glucose) not, and HB is for containing 10 ^-5The H-DMEM of M insulin; HC is for containing 10 ^-6The H-DMEM of M insulin; HD is for containing 10 ^-7The H-DMEM of M insulin.

Two kinds of rat blood sugar values of Fig. 3 compare * p＜0.05.Wistar rat and GK rat blood sugar measure by statistics that there is significant difference in Epidemiological Analysis, and keep steady statue always.

Fig. 4 GK rat tails blood glucose is in different times Fast Measurement result.G2 organizes before extraction and postoperative Quick Measuring rat tails blood glucose, and change of blood sugar is not obvious, does not have significant difference.

Fig. 5 A figure is Wistar rat exodontia x ray examination result during 4 week; B figure is the Wistar rat side 4 all x ray examination results that do not have tooth pulled out.

Fig. 6 IRmRNA not on the same group between the variation of exodontia nest healing 7d-28d, * p＜0.05, * * p＜0.01 and matched group ratio.WN group IR mRNA level shows as continuous decrease; GN group then shows as the IRmRNA level and begins trend G1 that dropping to raises first reduces afterwards organize and also show as afterwards reduction trend of first rising after 14d peaks, peak during 21d, horizontal G2 group is consistent with the performance of WN group when dropping to 7d subsequently when 28d, continuous decrease in the 7-28d process.

Fig. 7 IGF-1RmRNA not on the same group between the variation of exodontia nest healing 7d-28d, * p＜0.05, * * p＜0.01 and matched group ratio.The IGF-1R mRNA level of GN, G1 group is lower, and changes in this process without obvious significant difference; G2 group is at 7d, and 14d, 21d be apparently higher than other two groups, and reaches peak value when 21d; WN organizes 14d, and 21d is significantly higher than GN, G1 group (p＜0.01).

The specific embodiment

Embodiment 1 insulin is on the impact of In vitro culture normal rat mandibular bone osteoblastic proliferation

Present embodiment is take SPF level Wistar rat as material, and male and female are not limit, body weight 180-220g, age in 6-8 week.

Experimental technique and step

1) the disappear method that combines with piece of tissue of enzyme is cultivated adult rat mandibular bone osteoblast.

Get the Wistar rat, after disconnected neck is put to death, in 75% ethanol, soak 5min; In aseptic super-clean bench, rat is lain on the back, with povidone iodine cotton swab sterilization rat neck and oral cavity, cut off rat skin in cervical region, successively peel off, expose mandibular bone; Aseptic taking-up places 75% ethanol to wash 5-10sec with the mandibular bone of muscle and fascia, soaks 1min among the 5.25%NaClO, and PBS (containing 100IU/ml penicillin, 100IU/ml streptomycin) washes 3 times repeatedly; Pull out tooth, thoroughly strike off muscle, fascia and periodontal tissue; Mandibular bone is shredded osteocomma into about 2mm * 2mm with rongeur, and the PBS buffer repeatedly washes to the osteomiosis piece and is white in color; Osteocomma is inserted in the little culture bottle, add 0.125% trypsin 37C, 150rpm digestion 8min in the water bath chader, digest 6 times and stop afterwards; Collect the 2nd to 6 time Digestive system through 200 order stainless steel sift net filtrations and centrifugal, (the DMEM culture medium is DMEM powder 13.4g, adds NaHCO with the L-DMEM that contains 20% hyclone behind the removal supernatant ₃2g, two ionized water 1L that boil off transfer pH value to 7.0, filtration sterilization, 4 ℃ of preservations of packing.Include 100IU/ml penicillin, 100IU/ml streptomycin) re-suspended cell; Cell is inoculated in the culture bottle at 37 ℃, 5%CO ₂, in the constant temperature incubator, leave standstill under the saturated humidity condition and cultivate 40min; Differential velocity adherent is removed fibroblast, continues behind the purification osteoblast to cultivate

The piece of tissue of being left after enzymic digestion shreds again to 1mm * 1mm, is laid on to be inverted in the culture bottle to cultivate, and turns over bottle behind the 4h.Treating that cell covers with goes down to posterity.When going down to posterity, differential velocity adherent 40min is the purification osteoblast repeatedly.The cell of enzyme digestion and tissue mass cell culture all changes full liquid in 48h, changes half liquid or 1/3 every 48h later on and changes liquid.The lower tight observation of cell upgrowth situation of inverted microscope, 80% rear usefulness 0.125% pancreatin (containing 0.02%EDTA) had digestive transfer culture is cultivated at the bottom of cell covers with bottle.Get well-grown the 4th generation cell be used for experiment and cell function is identified.

2) mandibular organization's osteoblast is identified

With the 4th generation osteoblast be seeded on the coverslip, cultivate behind the 3w with the fixing 15min of 95% ethanol, carry out ALP with Gomori calcium-cobalt method and dye.Step is as follows: the coverslip that fixes is inserted Incubating Solution (3% sodium β-glycerophosphate 5ml, 2% pentobarbital sodium 5ml, 2% lime nitrate 10ml, 2% magnesium sulfate 5ml, distilled water 25ml after the distilled water rinsing.) in; Hatch 2h for 37 ℃, tap water flushing 3 times; Hatch 1min in 2% cobalt nitrate, the tap water flushing; Natural drying, sealing.

Get and be covered with in 6 orifice plates of coverslip at the bottom of the 4th subtituted culturing cell is inoculated in the hole, use instead in the L-DMEM culture fluid (wherein containing 50ug/ml vitamin C and 10mmol/L sodium β-glycerophosphate) that contains 10% new-born calf serum and cultivate, every 3d changes liquid once, in 21d, see under the mirror and take out coverslip when more opaque mineralising tuberosity is arranged, dye the calcium method by alizarin red S and dye.Step is as follows: culture dish is washed 2 times with PBS, and 95% ethanol original position is 10min fixedly; 1% alizarin red (2% alcohol, PH8.3) dyeing 5min; 50% ethanol is removed non-specific painted; Natural drying.

With the 4th generation osteoblast be seeded on the coverslip, cultivate behind the 3w with the fixing 15min of 95% ethanol, carry out type i collagen dyeing with Van Gieson method, step is as follows: plain of Garapa dyes 5min, and PBS washes 3 times; Blue 10min is returned in the tap water vibration; Picric acid acid fuchsin dyeing 5min; 95% ethanol breaks up fast; Ethanol dehydration, dimethylbenzene is transparent, the resin mounting; Optical microphotograph Microscopic observation and shooting.

3) immunohistochemical staining of cell climbing sheet

Make cell climbing sheet: with matched group and diabetic groups the 5th generation cell with 2 * 10 ⁴/ ml is inoculated on the slide, behind the cultivation 5d, and fixed cell.PBS (0.01M PBS contains 0.05% tween) washes 5min, puts into wet box; Drip 0.3%Triton X-100, place 37 ℃ of incubators to hatch 15min; Triton X-100 on the creep plate is dried, and PBS shakes and washes, 5min * 3 time; Drip 3%H ₂O ₂(0.1ml 30%H ₂O ₂With the abundant mixing of pure 100% methanol of 10ml, matching while using, 37 ℃ of incubators are hatched 20min; PBS shakes and washes, 5min * 3 time; Drip the antibody of an amount of dilution, IR α 1 and IGF-1R α 1 all diluted with 1: 100, and 4 ℃ are spent the night.37 ℃ of rewarming 1h, PBS shakes and washes, 5min * 3 time; Drip reagent one liquid in the SP test kit, place 37 ℃ of incubators to hatch 10min; PBS shakes and washes, 5min * 3 time; Drip reagent two liquid in the SP test kit, place 37 ℃ of incubators to hatch 10min; PBS shakes and washes, 5min * 3 time; Drip DAB nitrite ion (the 1ml distilled water adds 1 of concentrated DAB liquid, matching while using), control colour developing under the mirror; The distilled water color development stopping; Haematoxylin is redyed 2min, washing, and 1% hydrochloric acid solution (70% alcohol) the differentiation several seconds, mobile tap water returns blue 20min; Ethanol dehydration, dimethylbenzene is transparent, the resin mounting; The criterion of staining power: non-coloring in feminine gender-nucleus or the kytoplasm; Light yellow dyed particles appears in the weak positive-nucleus or the kytoplasm; The brown color dyed particles appears in positive cell nuclear or the kytoplasm; The dark-brown dyed particles appears in strong positive-nucleus or the kytoplasm.

4) select the insulin suitable concentration

Every group is established 12 samples is 2 * 10 with density ⁴The cell of individual/ml is inoculated in 96 orifice plates, is changed to serum-free medium behind the cell fusion, adds next day and contains 5% hyclone.The variable concentrations (0,10 of the culture fluid preparation of experiment grouping: 5.5mM glucose (5.5mMG) L-DMEM (physiology sugar concentration), 16.5mMG (high sugar) concentration H-DMEM ^-5M, 10 ^-6M, 10 ^-7M) insulin is cultivated, and number consecutively is LA, LB, LC, LD; HA, HB, HC, HD; Continue to cultivate 7d, the next day change medicine and culture medium; At the last 4h of cultivation cycle, every hole adds the MTT of 20 μ l, continues to hatch to end; Add DMSO, survey absorbance (OD) value in 490nm with microplate reader.Mtt assay detects osteoblastic propagation, determines the medicine optimum concentration.

Experimental result and discussion

1) osteoblast is identified

Alkaline phosphatase staining: behind the Growth of Cells 21d, be paved with whole coverslip fully, in the ALP dyeing visible cell matter a large amount of brown particles or block precipitation arranged.Mineralising tuberosity dyeing: osteoblast under β-phosphoglycerol sodium existence condition, can make the extracellular matrix mineralising at vitamin C, and forms volume mineralising tuberosity, through the Alizarin red staining block precipitation that takes on a red color.Type i collagen dyeing: ripe osteoblast can synthesize type i collagen, is one of osteoblast mark, the dyeing of the visible large tracts of land pink of collagen staining.IR and the IGF-1R expression on osteoblast.IR and IGF-1R all have expression on osteoblast, be uniformly distributed in the osteoblastic endochylema.

2) sugared concentration and insulin are for the impact of osteoblastic proliferation

Give respectively the variable concentrations (0,10 of the culture fluid preparation of 5.5mM glucose (5.5mMG) L-DMEM (physiology sugar concentration), 16.5mMG (high sugar) concentration H-DMEM ^-5M, 10 ^-6M, 10 ^-7M) insulin is cultivated, and number consecutively is LA, LB, LC, LD; HA, HB, HC, HD.Osteoblast is cultivated in two kinds of culture medium of 5.5mMG, 16.5mMG, and it is more active to see that osteoblastic propagation shows in L-DMEM, the osteoblastic propagation of high sugared surround inhibition.Figure 1 shows that insulin of different concentration is for the impact of osteoblastic proliferation, 10 in L-DMEM ^-6M and 10 ^-7The insulin of M concentration all promotes osteoblastic propagation.Fig. 2 is presented at that the insulin concentration difference is for the impact of osteoblastic proliferation among the H-DMEM, and osteoblast is 10 ^-6Propagation is more active in the M insulin concentration.

This experiment has been cultivated mandibular organization's osteoblast under two kinds of sugared concentration, found that the osteoblastic propagation of high sugared surround inhibition mandibular organization, and 10 ^-6M concentration insulin can promote the osteoblastic propagation of mandibular organization.Hyperglycemia changes the secretion of the insulin of glucose mediation, and this is called as glucose toxicity.The glucose of high concentration can affect insulin by a lot of approach, and glucose comprises many genes of IGF-1R and IR gene by adjusting, and is to affect the effect of insulin transcribing and translate two horizontal adjusted; The result that also can activate by serine phosphorylation and the tyrosine phosphatase of Profilin kinase c mediation reduces activity [the Berti L of IR, Mosthaf L, Kroder G et al.Glucose-induced translocation of protein kinase C isoforms in rat-1 fibroblasts is paralleled by inhibition of the insulin receptor tyrosine kinase.J Biol Chem.1994,269 (5): 3381-6.M ü ller HK, Kellerer M, Ermel B et al.Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells.Diabetes.1991,40 (11): 1440-1448.].

Embodiment 2 insulin gels promote GK rat exodontia nest Healing to mandibular organization's topical

Present embodiment is take Goto-Kakizaki (GK) rat as material, Goto-Kakizaki (GK) rat is that the people such as Goto in 1975 are at Japanese celestial platform, 18 slight impaired glucose tolerance Mus are selected in oral administration carbohydrate tolerance experiment (OGTT) from 211 Wistar rats, through about 10 generations, repeatedly select the copulation of hyperglycemia Mus, form the spontaneous nonobese type 2 diabetes mellitus Mus kind approximate with human type 2 diabetes mellitus, be called for short the GK rat.This Mus kind main manifestations is that the insulin secretion that stimulates of glucose is impaired, and the beta cell secretion is impaired, and hyperglycemia, hepatic glycogen generate and increase, long-term ill after, various complication appear.Type 2 diabetes mellitus can be divided into the absolute not enough and insulin relative deficiency of insulin, needs clinically the supplemented with exogenous insulin to treat.1980 and the WHO of World Health Organization (WHO) in 1988 are as follows about the diagnostic criteria of diabetes: diabetic symptom is arranged, possess following any one can be diagnosed as diabetes a, fasting glucose 〉=7.8mmol/l; B, blood glucose 〉=11.1mmol/l any time in a day; C, fasting glucose (7.8mmol/l, but oral 75% glucose tolerance test, two hours blood glucoses 〉=11.1mmol/l.Show that in a few studies in the past diabetics topical application some drugs can promote wound healing, these medicines comprise protease inhibitor and plasma fibronectin element etc.GK rat kind main manifestations is that the insulin secretion that stimulates of glucose is impaired, and the beta cell secretion is impaired, and hyperglycemia, hepatic glycogen generate and increase, long-term ill after, various complication appear.How the topical application insulin is expressed in this process the effect of diabetes exodontias nest healing and IR and two kinds of receptors of IGF-1R and is changed not yet clear and definite.This research is take the Wistar rat as contrast, the interior part of exodontia nest gives the insulin gel after pulling out GK rats with left mandibular incisors, the insulin topical application is on impact and the exodontia nest healing state of IR and IGF-1R, to reach experiment purpose in the observation exodontia nest agglutination.

Experimental technique and process

1) foundation of Wistar rat and GK rats ' mandible exodontia snap type

Random packet: Wistar rat exodontia group (WN); GK rat exodontia group (GN), GK rat injection chitosan gel rubber group (G1), GK rat are pulled out the chitosan gel rubber group (G2) that injection is loaded with insulin, 3 every group.Take by weighing body weight, afterbody blood glucose measurement and labelling are with rat femoribus internus lumbar injection 1% pentobarbital liquid (0.5ml/100g body weight); Fixing, sterilization, drape.Lower jaw left side incisor separates gingiva, does trapezoidal cut in alveolar bone lip side, opens words side mucoperiosteum lobe; Remove the lip side seam plate of small part, reduce the bone resistance of lip side; The tongue side is utilized gingival separator that tooth and alveolar bone are separated gently and is made needle holder can clamp the Rat Mandibular incisor to get final product; Tooth socket is processed: strike off periodontal tissue in the tooth socket, normal saline suitably after the flushing according to the different grouping nest local injection insulin gel of having tooth pulled out; Close wound: the para-position of trying one's best of the mucoperiosteum of a side and offside mucoperiosteum is tightly sewed up after will having tooth pulled out.Postoperative is measured rat tail blood blood glucose before with art; After rat is fully clear-headed, put back to the Mus cage; Intramuscular injection: benzylpenicillin sodium for injection 200,000 unit solution (0.1ml/100g); Amoxicillin Capsules is dissolved in three days (0.25mg/d) in the drinking bottle.The SPSS13.0 statistical software analyzes, and experimental data is used Expression, group difference relatively adopt One-Way ANOVO to analyze the LSD method, and P＜0.05 thinks that difference has significance.

2) the insulin gel promotes GK rat exodontia nest Healing to mandibular organization's topical

After preparing respectively sodium β-glycerophosphate solution and chitosan-acetic acid solution, use after being cooled to room temperature.Behind two kinds of solution ice baths, draw chitosan-acetic acid solution in container, rapid stirring dropwise adds sodium β-glycerophosphate solution simultaneously, and continues to stir, and obtains chitosan/sodium β-glycerophosphate hydrogel.Add an amount of insulin, preparation is to the insulin gel of mandibular organization's topical again.

1. draw materials and fixing

With the rat of experiment in one respectively at postoperative 7d, 14d, 21d, 28d draws materials.The grouping situation is with front portion experiment grouping.Mandibular bone is fixing outward: after separating mandibular bone, separate at the place of associating, center, clean out the muscle depended on, fascia, periodontal membrane after, in near take mandibular first molar as the boundary, mandibular bone is divided into front and back two parts, and it is frozen that the liquid nitrogen tissue is put into rapidly in the front portion, and it is fixing that 4% paraformaldehyde is put at the rear portion.Imaging examination: film making was observed each and is organized osseous tissue healing of exodontia nest and new bone formation situation after the x-ray inspection separated at the place of associating, center.

2. pathological examination

Be organized in fix 72 hours under the room temperature in the paraformaldehyde after, taking-up is put into the decalcification of 15%EDTA (pH=7.4) liquid and is processed, and dewaters paraffin embedding about 1 month after organizing decalcification fully, with 5 millimeters thickness uniformly slicings, save backup in the dry rear 4 ℃ of refrigerators of baking sheet machine baking sheet.

Dehydration and embedding step: gradient ethanol (70% ethanol 60min, 80% ethanol 60min, 90% ethanol 60min, 95% ethanol I30min, 95% ethanol II30min, 100% ethanol I20min, 100% ethanol II20min) dewaters step by step; Dimethylbenzene transparent (dimethylbenzene I and each 15min of dimethylbenzene II); 62 ℃ of waxdip 〉=3h, the routine paraffin wax embedding; Specimen is done the middle-distant direction Serial tissue sections by horizontal direction, the thick 5um of sheet; Choosing has the new osteogenetic tissue slice in exodontia nest zone, places on the microscope slide.

Paraffin organization section dewaxing step: dimethylbenzene I and the dimethylbenzene II 20min that respectively dewaxes; Gradient ethanol (100% ethanol 2min, 95% ethanol 1min, 80% ethanol 1min, 75% ethanol 1min, tap water flushing 5min, distilled water flushing 2min) is to washing.

Tissue slice carries out three groups of experiments: the conventional dimethylbenzene of cutting into slices, gradient ethanol dewax to water; Brazilwood extract dyeing is 3 minutes after the washing, the differentiation several seconds in 1% hydrochloride alcohol after the washing; Wash and return blue the immersion 30 seconds in rear 40 ℃ of water; Redyed 5 minutes in 0.5% Yihong after the washing; 70% ethanol, 85% ethanol, 95% ethanol, dehydrated alcohol dehydration; Dimethylbenzene is transparent; The neutral gum sealing.

Immunohistochemical method: detect IR and the IGF-1R expression in rats ' mandible incisor exodontia nest.Step is as follows: the paraffin section routine dewaxes to water; Blocking-up deactivating endogenous peroxydase: 3%H ₂O ₂Hatch 10min for 37 ℃, PBS washes 3 times * 5min; Antigen retrieval: 0.125% pancreatin (pH=7.2) carries out antigen retrieval 20min, and PBS washes 3 times * 5min; The sealing of normal serum working solution is hatched 10min, is inclined and do not wash for 37 ℃; Drip primary antibodie (IR=1: 100; IGF-1R=1: 100) 4 ℃ of refrigerator overnight incubation, 37 ℃ of rewarming 1h, 3 times * 5min of PBS flushing (replacing primary antibodie to make negative control with the PBS buffer); It is anti-to drip biotin labeling two, hatches 20min for 37 ℃, and PBS washes 3 times * 5min; Drip the streptomycin avidin working solution of horseradish peroxidase-labeled, hatch 20min for 37 ℃, PBS washes 3 times * 5min; DAB reacts dyeing, and colour developing is generally room temperature 3min, examines under a microscope the colour developing situation, tap water cessation reaction after the colour developing fully.After tap water fully washed, haematoxylin was redyed, and conventional dehydration is transparent, drying, mounting.ImmunohistochemistryResults Results is judged: the positive expression criterion is to observe to be centered around on the area of new bone girder osteoblast on every side in the endochylema whether the brown color electrodeposition substance is arranged, tinctorial strength is divided into 4 grades: colourless negative, faint yellow is the weak positive, and brown color is positive, and sepia is strong positive.

Goldner ' s three-color process colouring method: the 15min that in Weigert ' s Garapa element liquid, dyes of the section after the dewaxing, distilled water rinsing 1 time, tap water flushing 2 times; The 15min that dyes in 1% Ponceaux-acid fuchsin dyeing liquor, 1% acetate solution rinsing 2 times; 1% phosphotungstic acid-orange G 8min, 1% acetate solution rinsing 2 times; Viride nitens liquid dyeing 15min, 1% acetate solution rinsing 2 times; 70% ethanol rinsing 1 time, dehydrated alcohol I, II rinsing 2 times; Dimethylbenzene is transparent; The neutral gum sealing.

3. the processing of mandibular organization's osteocyte

Total RNA extracts: will be organized in the liquid nitrogen and grind, every 50-100mg tissue adds 1mlTrizol, carries out homogenized with Syrup-homogenizing instrument.Sample volume should not surpass Trizol volume 10%; The homogenate sample is placed 5min in room temperature (15-30 ℃), the nucleic acid-protein complex is separated fully; Every use 1mlTrizol adds the 0.2ml chloroform, thermal agitation 30s, and room temperature is placed 3min; The 2-8 ℃ of centrifugal 15min of 10000 * g.Sample is divided into 3 layers; Upper water is moved on in the new pipe mutually, with the RNA of isopropanol precipitating aqueous phase.Every 1ml Trizol adds the 0.6ml isopropyl alcohol, and room temperature is placed 10min; The 2-8 ℃ of centrifugal 15min of 10000 * g removes supernatant; Precipitate with 75% washing with alcohol RNA.2-8 ℃ is no more than the centrifugal 5min of 7500 * g, abandons supernatant; Room temperature is placed dry RNA precipitation.Add 30 μ l DEPC H ₂O dissolves RNA.-70 ℃ of preservations.

The RNA reverse transcription is cDNA, carries out afterwards the quantitative fluorescent PCR monitoring experiment, and the reaction system of quantitative fluorescent PCR is by 2 * SYBR Real-Time PCR mix, special primer, Taq enzyme, cDNA template, dd H ₂O forms.Fluorescent labeling is: 5 ' end FAM, it is confidential reference items albumen that 3 ' end TAMRA selects GAPDH.The specific design of primer is as follows:

GAPDHForward 5′-GAAGGTGGAGGTCGGAGTC-3′

GAPDH Reverse 5′-GAAGATGGTGATGGGATTTC-3′

IR Forward 5′-AGGAGCCCAATGGTCTGA-3′

IR Reverse 5′-GAGACGCAGAGATGCAGC-3′

IGF-1R Forward 5′-CGATGTGTGAGA AGACCACCA-3′

The SPSS13.0 statistical software analyzes, and experimental data is used

(One-Sample T Test, Paired-Sample T Test) checked in expression, group difference relatively user's difference analysis (One-Way ANOVA), T.

Experimental result and discussion

1) foundation of Wistar rat and GK rats ' mandible exodontia snap type

The interior rat of postoperative 2 ± 0.5h is all clear-headed, can freely movablely in mouse cage also take food after regaining consciousness, and from the postoperative to 7d, 14d, 21d, 28d, 12 Wistar rats are in good condition; 36 GK rats are all clear-headed and in good condition immediately after surgery, until draw materials (time of drawing materials is organized with Wistar).Wistar rat and GK rat body weight compare, and the Wistar rat body weight all overweights the GK rat, and both have significant difference P＜0.05 (table 1) statistical analysis.

Two kinds of rats of table 1 are the weight ratio (x ± s) of postoperative in the preoperative

* p＜0.05 is compared with matched group

Before the Rhizoma Atractylodis Macrocephalae, postoperative 30min gets WN group and G2 group rat tails blood glucose is measured, relatively the part gives change of blood sugar behind the insulin.Wistar rat and GK rat blood sugar measure by statistics that there is significant difference in Epidemiological Analysis, and keep steady statue (Fig. 3) always.G2 organizes before extraction and postoperative Quick Measuring rat tails blood glucose, and change of blood sugar is not obvious, does not have significant difference.Under two kinds of same diet feeding conditions of rat, GN, G1, G2 group rat is until still remain on hyperglycemia when drawing materials, apparently higher than WN group (Fig. 4). detection blood glucose value Wiatar group and GK group have obvious significant difference in the rat feeding process, the GK rat remains on hyperglycemia state always, before the extraction, the experimental group (G2) that postoperative is loaded with the chitosan gel rubber system of insulin for exodontia nest injection carries out rat tail blood blood glucose Fast Measurement and finds that change of blood sugar is little, no difference of science of statistics, illustrate that insulin is in the chitosan gel rubber system, directly do not enter blood by the exodontia nest and regulate rapidly blood glucose, raising is identical with Wistar with eating condition, the GK rat all remains on hyperglycemia state during time point until draw materials, and meets this requirement of experiment.

It is 10 that the administration slow-released system contains concentration ^-6M-10 ^-7The chitosan of the insulin of M, percentage by weight 2.5%-3.0% and the sodium β-glycerophosphate of 5.0%-6.0%, wherein pH value is 6.9-7.2.

Gross examination: the equal first phase healing of four groups of Rat Mandibular incisors of postoperative 7d gross examination of skeletal muscle wound, mucosa is complete, and color and luster is normal, and same observation period experimental group and the healing of matched group wound surface show no obvious abnormalities, and healing is good, has no infection; Postoperative 28d observes substantially healing of wound.The x-ray check result: x-ray observation is 28d after two kinds of rat exodontias, can see the boundary that tooth socket is arranged on x line sheet, in the Wistar group tooth socket new bone formation is arranged obviously; Integral body has no obviously new bone formation in the GK group tooth socket, and the bone texture is compared disorder (Fig. 5) with the Wistar group.

The Wistar group: be flooded with hemocyte and early stage granulation tissue in the exodontia nest during exodontia 7d, granulation tissue wherein comprises blood vessel, fibroblast and chronic inflammatory cells; 28d after the exodontia, the area of new bone girder is obvious, and structure is also relatively more disorderly, not yet reconstruction.The GK group: during the rear 7d of exodontia, GN group inflammatory cell is compared inflammatory cell showed increased, fibroblast arrangement disorder with the WN group; G1 group inflammatory cell infiltrates more in granulation tissue, and the arrow place is C/GP among the figure; G2 group inflammatory cell is compared with the WN group and is increased, but compares to some extent minimizing with the G1 group, have no the structure such as G1 group C/GP in the staining section, but arrow is depicted as foreign-body giant cell among the figure, and representing has C/GP near it.28d after the exodontia, GN can see cube in organizing, polygonal osteoblast increases, and is obviously active near osseous tissue place osteoblast, begins to have new osteogenesis; Knitting is bad in the G1 group, and the fibroid sclerotin is main, and fibroblast, osteoprogenitor cell mix, and are mingled with indivedual inflammatory cells therebetween; Occur irregular bone substrate and the area of new bone girder of a little in the G2 group, the osteoblast around the area of new bone girder is obviously active, and cube, polygonal osteoblast are common.

Goldner ' s three-color process result: because HE dyeing can not be distinguished the stage of bone mineralising fully, general Goldner ' the S three-color process that adopts, carry out paintedly mainly for collagen fiber, and active osteoblast cell space is cube or doll shape, and kytoplasm is strong basophilia.

The expression of IR and IGF-1R: IR and IGF-1R receptor are all expressed on osteoblastic endochylema, in the visible endochylema of the cell of positive expression the brown color electrodeposition substance are arranged.Because this section is the paraffin section that decalcification is processed, so the tissue after the decalcification has the cell cytosol dissolving the non-specific dyeing of small part to occur, the criterion of this experiment positive expression is to observe to be centered around on the area of new bone girder osteoblast on every side in the endochylema whether the brown color electrodeposition substance is arranged.Can see the weak positive expression of IR in a small amount of freshman bone tissue during G2 group 28d, GN group 28d then is the IR positive expression; IGF-1R positive expression on osteoblast during WN group 28d around new life's the bone trabecula, the GN that organizes with the time organizes 28d there are no the area of new bone girder, the weak positive expression of IGF-1R.

Real-time qPCR result: on rat exodontia nest 7-28d agglutination IRmRNA level, WN group IR mRNA level shows as continuous decrease; GN group then shows as the IRmRNA level and begins trend G1 that dropping to raises first reduces afterwards organize and also show as afterwards reduction trend of first rising after 14d peaks, peak during 21d, horizontal G2 group is consistent with the performance of WN group when dropping to 7d subsequently when 28d, continuous decrease in the 7-28d process (Fig. 6).The IRmRNA level the highest (p＜0.01) of GN group wherein, G1, G2 group is close with the WN group all to be lower than the GN group.On rat exodontia nest 7-28d agglutination IGF-1RmRNA level, the IGF-1R mRNA level of GN, G1 group is lower, and changes in this process without obvious significant difference; G2 group is at 7d, and 14d, 21d be apparently higher than other two groups, and reaches peak value when 21d; WN organizes 14d, and 21d is significantly higher than GN, G1 group (p＜0.01) (Fig. 7).

Find among the present invention when Wistar group exodontia 7d, to be flooded with hemocyte and early stage granulation tissue in the exodontia nest, wherein comprise blood vessel, fibroblast and chronic inflammatory cells; 28d after the exodontia, the area of new bone girder is obvious, and structure is also relatively more disorderly, not yet reconstruction.The pathological change that GK group 28d organizes except G2 changed when being similar to WN group 28d, the rear 28d fibroid sclerotin of all the other group exodontias was main, and fibroblast, osteoprogenitor cells mix, and are mingled with indivedual inflammatory cells therebetween.Goldner ' s three-color process result confirmed to observe among the HE result shown in the area of new bone trabecularism.Because being decalcification, this section processes rear paraffin section, so the decalcification tissue has part cell plasmolysis the non-specific dyeing of small part to occur.The immune group chemistry observes IR and the IGF-1R receptor is all expressed on osteoblastic endochylema, and IR can see weak positive expression when G2 group 28d, then be positive expression during GN group 28d; Positive expression on the osteoblast around IGF-1R new life's when WN group 28d the bone trabecula, the weak positive expression of IGF-1R during GN group 28d.It also is that area of new bone forms active place that IGF-1R expresses active place at osteoblast.Utilize Real-time qPCR method to detect respectively level and the variation of rat exodontia nest 7-28d agglutination IRmRNA and IGF-1RmRNA, discovery is for IRmRNA, G2 group variation tendency is organized the consistent downward trend that is with WN, the level of GN group is the highest simultaneously, and has the trend that raises first and reduce afterwards; In this process for the IGF-1RmRNA level, the level of G2 group is significantly higher than other GN, two groups of G1, to organize the IGF-1R mRNA level of GN, G1 group in fact lower but be lower than WN, and change in this process without obvious significant difference.The gel systems local injection of insulin slow release does not change rat blood sugar at once in rat exodontia nest; The insulin nest that is applied topically to have tooth pulled out has changed IR and IGF-1R level, has promoted that area of new bone forms in the diabetes rat exodontia nest.

Claims

1. an insulin topical gel systems is characterized in that, it is 10 that described gel systems contains concentration ^-6M-10 ^-7The chitosan of the insulin of M, percentage by weight 2.5%-3.0% and the sodium β-glycerophosphate of 5.0%-6.0%, pH value are 6.9-7.2.

2. insulin topical gel systems claimed in claim 1 promotes purposes in the medicine of mandibular organization's healing in preparation, and described medicine is locally applied to animal live soma.

3. purposes according to claim 2 is characterized in that, described animal live soma is animal mandibular organization.

4. purposes according to claim 3 is characterized in that, described animal mandibular organization refers to that mandibular organization is at the osteoblast of In vitro culture and in the mandibular organization of living animal.