CN103566413A - Thermo-sensitive gel composition and application thereof - Google Patents
Thermo-sensitive gel composition and application thereof Download PDFInfo
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- CN103566413A CN103566413A CN201310522096.3A CN201310522096A CN103566413A CN 103566413 A CN103566413 A CN 103566413A CN 201310522096 A CN201310522096 A CN 201310522096A CN 103566413 A CN103566413 A CN 103566413A
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Abstract
The invention relates to a thermo-sensitive gel composition. The composition comprises poloxamer segmented copolymer and indocyanine green. A thermo-sensitive gel water solution prepared from the thermo-sensitive gel can be prepared into an intravascular marked disrupting agent which has an excellent bio-compatibility and no toxic or side effect, can temporarily disrupt a target hepatic segment portal venous flow and realize accurate location of hepatic segment and blood vessels. The preparation can be used in clinical application of surgical operations, enables a resection part to be easily identified in an operation while performing disruption, realizes accurate location of the operation, and can greatly reduce the bleeding amount, so that a doctor can complete a resection operation more easily and more safely by means of the preparation, and the operation safety can be improved. The composition is arranged in a kit for preparing the thermo-sensitive gel water solution, can be stored and transported conveniently, and can be used for conveniently preparing the marked disrupting agent for surgical operations. The kit is easy to operate, low in cost and safe and convenient to use.
Description
Technical field
The invention belongs to thermo-responsive hydro gel preparation and medical applications technical field thereof, be specifically related to a kind of thermo-responsive hydro gel compositions and the application in surgical operation thereof.
Background technology
At present surgical operation enters " precisely " epoch, and desirable RESECTION OF LIVER CANCER is not only answered complete removal of lesions was, this hepatic segments that also should removal of lesions was place.Yet the liver of human body is a whole substantial viscera in appearance, according to its internal blood vessel distribution and supply of blood flow, can be divided into artificially 8 sections, realize the key of accurate hepatic segments/sub-hepatic segments excision demarcates target hepatic segments or sub-liver section boundary in Rhizoma Atractylodis Macrocephalae, realize the key of accurate hepatic segments/sub-hepatic segments excision demarcates target hepatic segments or sub-liver section boundary in Rhizoma Atractylodis Macrocephalae, in order to divide accurately a certain liver section boundary in operation process, at present clinical conventional method has following three kinds: (1) is by conjunction with liver surface anatomic landmark with intraoperative ultrasound is followed the trail of hepatic vein and the projection of branch of portal vein on liver surface decides target liver section boundary.The method is simple to operate, but requires dissecting and have enough knowledge in liver, and skillfully grasps intraoperative ultrasound.The specimen of often adopting excision is in this way all regular shape, is not section or sub-section excision truly, only reaches degree relatively accurately; (2) by dissecting porta hepatis, and block after dissecting the Glisson base of a fruit of free target hepatic segments in liver parenchyma, realize block completely target hepatic segments blood for after, along ischemia border, realize hepatic segments and excise.Yet realize that porta hepatis is dissected and to the Glisson base of a fruit of free section in liver parenchyma difficulty comparatively, specification requirement is high, risk is larger, has limited the universal of this technology; (3) ultrasonic guidance portal vein puncture injection stain: first it proposed by professor Makuuchi, is most widely used at present.After intraoperative ultrasound Conducted Puncture branch of portal vein, inject methylene blue, target hepatic segments surface generation indigo plant is dyed immediately, dyes border can complete hepatic segments/sub-hepatic segments excision along indigo plant.Though the method blemish in an otherwise perfect thing has been to realize the target on accurate demarcation hepatic segments border, during still from disconnected liver parenchyma, mostly still need to adopt Pringle method blocking-up porta hepatis, unavoidably liver is caused to ischemical reperfusion injury.Said method none there is not complicated operation, risk is larger or the shortcoming such as expensive, therefore a lot of surgeon remain not accurately " excavate part " while carrying out hepatectomy at present.
Therefore, the problem existing is at present need to research and develop a kind ofly to have good biocompatibility, have no side effect, can temporary interruption target hepatic segments portal venous flow, and realize the blood vessel internal labeling surgical operation preparation of hepatic segments and blood vessel precise positioning.
Summary of the invention
Technical problem to be solved by this invention is for above-mentioned the deficiencies in the prior art, a kind of thermosensitive hydrogel compositions being comprised of poloxamer block copolymer and indocyanine green is provided, the thermosensitive hydrogel composition solution being made by this thermosensitive hydrogel compositions has the feature of temperature sensitivity, reversible, nontoxic and labelling, for surgical operation, can play good blocking-up and labelling effect, can help surgeon to complete more simple, the safer resection operation of hepatic segments accurately.
The present invention also provides a kind of above-mentioned thermosensitive hydrogel aqueous solution in the application for the preparation of in operating labelling blocking-up preparation.
For convenience of using, the present invention also further provides the test kit that contains thermosensitive hydrogel compositions.
For this reason, first aspect present invention provides a kind of thermosensitive hydrogel compositions, comprises poloxamer block copolymer and indocyanine green, and wherein, the mass ratio of poloxamer block copolymer and indocyanine green is 640: 1～800: 1.Preferably the mass ratio of poloxamer block copolymer and indocyanine green is 720: 1.
According to the present invention, described poloxamer block copolymer is polyoxyethylene-poly-the third ethylene-polyoxyethylene triblock copolymer, and wherein polyoxyethylene is 7: 3 with the mol ratio of poly-the third ethylene.For example, described poloxamer block copolymer is Poloxamer407(poloxamer188), its mean molecule quantity is 12000 dalton.
Second aspect present invention provides a kind of thermosensitive hydrogel aqueous solution that contains above-mentioned thermosensitive hydrogel compositions, and wherein, the content of poloxamer block copolymer is 16%～20%(weight/volume).Preferably the content of poloxamer block copolymer is 18%(weight/volume).
In the present invention, described water is sterile distilled water.
Poloxamer block copolymer has reversible temperature-sensing property, its can monomer form dissolve, surpassing CMC(critical micelle concentration) and CMT(critical micell temperature) time, its hydrophobic chain polyoxypropylene section dehydration polymerization becomes micelle, these spherical micelle kernels are polyoxypropylenes, outside is the polyoxyethylene chain that hydration is expanded, and continues to heat up, and micelle ordered arrangement forms solid gel.And lower than CMC(critical micelle concentration) and CMT(critical micell temperature) time poloxamer block copolymer again self-dissolving form solution.
For example, poloxamer188 (Poloxamer407) also has reversible temperature-sensing property.Compare with other several poloxamers, the concentration that poloxamer188 forms gel is lower, and the concentration that forms gel in the time of 25 ℃ is 20%(weight/volume).Poloxamer407 concentration is higher, and gel viscosity is higher.Gelation temperature is lower, and adhesion power is stronger, and after giving enough low temperature and sufficient equilibration time, the apparent viscosity of Poloxamer407 can recover.From above-mentioned, Poloxamer407 aqueous solution is in a liquid state while having low temperature, and temperature raises and changes solid gel into, and temperature transfers liquid reversible temperature-sensing property again to while reducing.And, through clinical trial, confirm that biochemical reaction does not occur in vivo for it, can, through urine with the metabolism of original shape form, human body be had no side effect.But this Poloxamer407 aqueous solution is water white, does not possess the effect of labelling or indication in operation process, cannot realize the precise positioning of operation.
Above-mentioned characteristic based on poloxamer block copolymer, the inventor attempts Poloxamer407 as a kind of reversible blocker for surgical operation, for making its performance that has labelling concurrently and then realizing the function of precise positioning and blocking-up in operation, trial adds Poloxamer407 aqueous solution by indocyanine green, yet the influence factor of Poloxamer407 critical micell temperature is more, as homologue effect, saltout and salting in effect etc., after adding ICG, its critical micell temperature may also can change, therefore be necessary to study the critical micell temperature of P407-ICG thermosensitive hydrogel aqueous solution, dissolution characteristics, stability, the results are shown in Table 1:
The characteristic of table 1Poloxamer407-indocyanine green thermosensitive hydrogel aqueous solution
| Concentration/%(weight/volume) | Critical micell temperature/℃ |
| 16 | 29 |
| 18 | 25 |
| 20 | 22 |
As can be seen from Table 1, this Poloxamer407-indocyanine green thermosensitive hydrogel aqueous solution therein concentration of Poloxamer407 is 18%(weight/volume) time at ambient temperature for green transparent solution, when environment temperature reaches more than 25 ℃, for example, under animal and human's body body temperature condition, just may be transformed into solid phase gel state, and when temperature is during lower than animal and human's body body temperature, its again self-dissolving become solution, it enters after blood circulation because concentration is far below the required 15%(weight/volume of gelling occurs again), so can not form again solid gel.The Poloxamer407 aqueous solution that this contains indocyanine green has no side effect, can prototype form through homaluria.Utilize this characteristic, can be by this Poloxamer407 aqueous solution that contains indocyanine green for making labelling blocking-up preparation, for the enforcement of assisted surgery.
Given this, third aspect present invention provides a kind of above-mentioned thermosensitive hydrogel aqueous solution in the application for the preparation of in operating labelling blocking-up preparation.
In the present invention, described surgical operation comprises accurate hepatic segments resection operation or sub-hepatic segments resection operation.
In the present invention, by cold cut, prepare thermosensitive hydrogel aqueous solution, wherein indocyanine green can be added to the water preparation thermosensitive hydrogel aqueous solution with poloxamer block copolymer simultaneously, also can be after being mixed with poloxamer block copolymer aqueous solution, then add wherein indocyanine green mix homogeneously to be mixed with thermosensitive hydrogel aqueous solution.
Prepared thermosensitive hydrogel aqueous solution or poloxamer block copolymer aqueous solution are placed under cryogenic conditions and are used after cold preservation.Described cryogenic conditions is 2～6 ℃, is preferably 4 ℃.The time of described cold preservation is 5～10 minutes, is preferably 5 minutes.
In one embodiment of the invention, when above-mentioned labelling blocking-up preparation being injected in portal vein in rats to (37 ℃ of body temperature), its gelling at once and become solid-state realize labelling and blocking-up in art, after about 10 minutes, it is spontaneous dissolving in blood vessel, formed solution enters after blood circulation because concentration is far below the required concentration 15%(weight/volume of gelling occurs again), so can not form again solid phase gel.There is not any biochemical reaction in this product, has good biocompatibility in vivo, have no side effect, finally with prototype form through homaluria.Because from as green, be convenient to very much identification in operation, can realize operation precise positioning simultaneously, greatly improve the safety of operation.
In another specific embodiment of the present invention, hepatic segments resection operation by above-mentioned labelling blocking-up preparation for Landrace: use ultrasonic guidance above-mentioned labelling blocking-up preparation to be injected to the blood vessel of target hepatic segments, this above-mentioned labelling blocking-up preparation is become solid-state at once from liquid state, blocked blood flow, ischemia border can be seen in corresponding hepatic segments surface, can complete immediately hepatic segments resection operation accurately; In disconnected liver parenchyma process, because vascular inner gel presents green, effectively guide the direction of the disconnected liver of doctor, further improved the safety of operation.
For convenience of using, fourth aspect present invention provides a kind of test kit of above-mentioned thermosensitive hydrogel compositions, the pad that it comprises lid, box body and is arranged at box body inside, pad surface is provided with four cavitys, wherein, the first reagent bottle containing poloxamer block copolymer is housed in the first cavity, the second reagent bottle containing indocyanine green is housed in the second cavity, the 3rd cavity is equipped with solution preparation device, and the 4th cavity is equipped with syringe.
According to the present invention, in described the first reagent bottle, in contained poloxamer block copolymer and the second reagent bottle, the mass ratio of contained indocyanine green is 640: 1～800: 1, is preferably 720: 1.
In one embodiment of the invention, solution preparation device is graduated bottle, and its scale capacity is counted 5～6.25ml/g with the quality of contained poloxamer block copolymer in the first reagent bottle, is preferably 5.56ml/g.
By the thermosensitive hydrogel aqueous solution of the formulated poloxamer block copolymer of the gel combination of poloxamer block copolymer of the present invention and indocyanine green and indocyanine green, can make labelling blocking-up preparation and can realize labelling blocking-up and the precise positioning of operation for surgical operation, greatly improve the safety of operation.
Especially poloxamer188-indocyanine green thermosensitive hydrogel aqueous solution, it is green transparent solution at ambient temperature, when environment temperature reaches more than 25 ℃, just can be transformed into solid phase gel state, through spontaneous dissolving in about 10 minutes, it enters after blood circulation because concentration is far below the required 15%(weight/volume of gelling occurs again), can not form again new gel.The labelling blocking-up preparation made from this thermo-responsive hydro gel composition solution has temperature sensitivity, feature reversible and labelling is nontoxic, and particularly this labelling blocking-up preparation has no side effect, can prototype form through homaluria.
The huge advantage that has that this labelling blocking-up preparation is used for the treatment of in hepatocarcinoma is: (1) meets the principle of tumor radical cure: the micrometastasis of sending out in the section of tumor and first generation is excised simultaneously simultaneously, reduce postoperative recurrence; (2) reduce intraoperative hemorrhage: in hepatic segments border, there is no the large Glisson base of a fruit three anatomical structures, from disconnected liver parenchyma, by relative depletion of blood area within a jurisdiction face, can reduce intraoperative hemorrhage; (3) reduce incidence of complications: owing to can not destroying trunk and bile duct, thereby greatly avoided bile leakage after operation, residual liver to occur ischemia or congestion and then caused the generation of the complication such as hepatic insufficiency; (4) reduce in hepatoma carcinoma cell art and send out the risk of transfer through portal vein adverse current.
This labelling blocking-up preparation can be played to good blocking-up and labelling effect for surgical operation.Use it for clinically, can in blocking-up, make band excision position be easy to recognize in art, realize the precise positioning of operation, can greatly reduce amount of bleeding, and avoid gallbladder in art to leak or sick cell migration simultaneously.Thus, can help surgeon to complete more simple, safer resection operation, improve operation safety.
The present composition is loaded in a kind of test kit of particular design, can conveniently store, transport the present composition, facilitate simultaneously the present composition is mixed with to labelling blocking-up preparation for surgical operation, easy and simple to handle, with low cost, safe and convenient to use.
Accompanying drawing explanation
Fig. 1 is the operating time schematic diagram in embodiment 2; Reference numeral in figure: SH organizes 144.2 ± 10.2min; PH organizes 98.3 ± 12.9min; P<0.05.
Fig. 2 is amount of bleeding schematic diagram corresponding with operating time in embodiment 2; Reference numeral in figure: SH organizes 83.3 ± 14.0ml; PH organizes 119.2 ± 18.6ml; P<0.05.
Fig. 3 is two groups of liver functions (glutamate pyruvate transaminase, ALT) change curves in embodiment 2.
Fig. 4 is two groups of liver functions (glutamic oxaloacetic transaminase, GOT, AST) change curves in embodiment 2.
Fig. 5 is two groups of renal functioies (blood urea nitrogen, BUN) change curves in embodiment 2.
Fig. 6 is two groups of renal functioies (serum creatinine, Scr) change curves in embodiment 2.
Fig. 7 is two groups of blood fat (triglyceride, TG) change curves in embodiment 2.
Fig. 8 is two groups of blood fat (T-CHOL, TC) change curves in embodiment 2.
Fig. 9 is the postoperative total bilirubin of Landrace (Tbil) change curve in embodiment 2.
Figure 10 is the postoperative Glutamyl transpeptidase of Landrace (γ-GT) change curve in embodiment 2.
Figure 11 is test kit schematic diagram of the present invention.
The specific embodiment
Below in conjunction with embodiment, describe the present invention in detail, these embodiment only play illustrative effect, are not limited to range of application of the present invention.Agents useful for same of the present invention or utensil are conventional medical agent or utensil except special instruction.
Of the present invention for preparing the test kit of thermosensitive hydrogel aqueous solution, the pad 3 that it comprises lid 1, box body 2 and is arranged at box body 2 inside, pad 3 surfaces are provided with four cavitys, wherein, the first reagent bottle 101 containing poloxamer block copolymer is housed in the first cavity 11, the second reagent bottle 102, the 3rd cavity 13 containing indocyanine green is housed in the second cavity 12 to be equipped with solution preparation device 103, the four cavitys 14 syringe 104 is housed.
In mentioned reagent box, in described the first reagent bottle 101, in contained poloxamer block copolymer and the second reagent bottle 102, the mass ratio of contained indocyanine green is 640: 1～800: 1, is preferably 720: 1.
In mentioned reagent box, solution preparation device 103 is thin mouthful graduated bottle, and its scale capacity is counted 5～6.25ml/g with the quality of contained poloxamer block copolymer in the first reagent bottle 101, is preferably 5.56ml/g.
In a preferred implementation, in the first reagent bottle 101, contain poloxamer block copolymer 18g; In the second reagent bottle 102, contain indocyanine green 25mg; Solution preparation device 103 is thin mouthful graduated bottle, and its scale capacity is 100ml.
In another preferred implementation, in the first reagent bottle 101, contain poloxamer block copolymer 9g; In the second reagent bottle 102, contain indocyanine green 12.5mg; Solution preparation device 103 is thin mouthful graduated bottle, and its scale capacity is 50ml.
Adopt mentioned reagent box to prepare thermosensitive hydrogel aqueous solution by cold cut, wherein indocyanine green can be added to the water preparation thermosensitive hydrogel aqueous solution with poloxamer block copolymer simultaneously, also can be after being mixed with poloxamer block copolymer aqueous solution, then add wherein indocyanine green mix homogeneously to be mixed with thermosensitive hydrogel aqueous solution.
Prepared thermosensitive hydrogel aqueous solution or poloxamer block copolymer aqueous solution are placed under cryogenic conditions and are used after cold preservation.Described cryogenic conditions is 2～6 ℃, is preferably 4 ℃.The time of described cold preservation is 5～10 minutes, is preferably 5 minutes.
Embodiment
Embodiment 1: portal vein in rats injection experiment
1. reagent and instrument
Reagent: poloxamer188 (P407, German BASF AG); Indocyanine green (ICG, Dandong Yi Chuan Pharmaceutical Co., Ltd).
Instrument: heat collecting type constant-temperature heating magnetic stirring apparatus (Beijing Medical Equipment Plant); 501 type thermostatic water-circulator bath instrument (Beijing Medical Equipment Plant); Laser Perfusion Imaging instrument (Britain Moor equipment company limited); Animal surgery operating theater instruments (Shanghai Medical Apparatus and Instruments Factory).
2. laboratory animal
10 of healthy male SD rats, body weight 240-260g ，You Military Medical Science Institute Experimental Animal Center provides.Ad lib water, illumination in 12 hours, starts experiment after adapting to one week.
3. the preparation of Novel temperature-sensitive gel and the investigation of gelation temperature
(1) P407 solution employing cold process preparation: take respectively 16%(weight/volume), 18%(weight/volume), 20%(weight/volume) three required P407 of variable concentrations solution, under magnetic agitation, gradation adds in the glass cillin bottle that distilled water is housed, 4 ℃ of ice-water baths stir it are uniformly dispersed, in the ratio of every 100ml solution 25mg, add ICG to carry out painted, then put 4 ℃ of refrigerator cold-storages and spend the night, the abundant swelling of gel is uniformly dispersed and obtains bottle-green P407-ICG thermosensitive hydrogel aqueous solution.
(2) appropriate P407-ICG thermosensitive hydrogel aqueous solution is put into glass cillin bottle, put it in ice bath, in the middle of the rubber stopper of bottle, punching and inserting precision is the precision thermometer of 0.1 ℃, and the mercury ball of thermometer submerges in gel solution completely.By magnetic stir bar, stir gel solution, make solution internal temperature even, simultaneously slow rising bath temperature, programming rate is about 1 ℃/min., glass cillin bottle is constantly tilted 90 °, the temperature when solution of take does not occur to flow is gelation temperature meanwhile.METHOD FOR CONTINUOUS DETERMINATION 3 times, getting its meansigma methods is gelation temperature.
4. blocking door vein effect research
(1) animal is fully conformed, fasting in preoperative 12 hours, can't help water.With after ether inhalation anesthesia success, rat dorsal position is fixed on wooden operating board, use iodophor disinfection.Get Median incision on upper abdomen and enter abdomen, otch is about 1.5cm, with Self-made hook, otch stomach wall is pulled open to both sides, with aseptic cotton carrier, gastrointestinal is dialled to caudal ward, fully appears hepatic portal structure.
(2) 9 SD rats of experimental group are divided into 3 subgroup A, B, C at random, 3 animals of each subgroup.With 1ml syringe, extract P407-ICG solution, in the main portal vein that accurately punctures is backward, inject 0.5ml, the solution concentration that A, B, tri-subgroups of C are injected is respectively 16%, 18%, 20%, observes Xining.1 rat of matched group adopts vascular clamp that portal vein is blocked separately.Adopt laser Perfusion Imaging instrument to monitor the perfusion situation of all rat blocking-up front and back liver, in order to observe the barrier effect of each group.
(3) under direct-view, observe main portal vein, wait for that it leads to and uses and use laser Perfusion Imaging instrument to confirm to close abdomen after liver perfusion returns to the front level of blocking-up.
(4) postoperative ad lib water, observes its long term survival rate and complication.
Its gelation temperature of P407-ICG solution that cold process prepares 16%, 18%, 20% concentration is respectively 29,26 and 22 ℃, in Table 2.The postoperative equal long-term surviving of all rats, has no any complication; After experimental group A, B, C and the blocking-up of matched group portal vein, rat liver perfusion all significantly declines, and the monitoring of laser Perfusion Imaging instrument shows perfusion value decline average out to 144PU, 148PU, 145PU and 131PU, in Table 2; The inner blocking-up of experimental group time limit result of study: A subgroup (16% gel solution) Xining 5min, B subgroup (18% gel solution) Xining 12min, C subgroup (20% gel solution) Xining 15min, in Table 2; After surpassing Xining, three subgroup rat liver perfusion situations all return to the front level of blocking-up.
Table 2 variable concentrations P407-ICG solution gel concentration and blocking-up rat portal vein time limit
Can find out, this experiment adopts that cold cut has prepared 16%, 18%, the P407-ICG thermosensitive hydrogel aqueous solution of 20% 3 kind of variable concentrations, result shows that its gelation temperature is respectively 29 ℃, 26 ℃ and 22 ℃, being injected in portal vein in rats (36.5 ℃) can form at once stable green gel and block into hepatic blood flow, use laser Perfusion Imaging instrument monitoring liver perfusion level to find, they are identical with traditional bulldog clamp blocking-up method effect.In experimentation, it is higher that we know from experience solution concentration, the resistance of injecting is larger, the time of blocking-up is longer, but the P407-ICG solution gel temperature that concentration is 20% is 22 ℃, this has all brought certain problems to temperature sensitive hydrogel storage and use, although and under 16% solution room temperature, can keep liquid (gelation temperature is 29 ℃), but because approaching critical gelling concentration (15%), its concentration causes the too short deficiency of Xining (only having 5min), therefore we to select concentration be that 18% P407-ICG solution carries out next step large zoopery, so not only solution at room temperature can keep liquid, be convenient to store and use, also blocked comparatively desirable duration simultaneously, be convenient to subsequent procedures operation.The equal long-term surviving of rat after this experimentation, has no any complication etc. abnormal, and this result is consistent with other bibliographical informations, has proved the safety of P407-ICG thermosensitive hydrogel aqueous solution through introportal infusion.
Embodiment 2: the hepatic segments excision experiment of Landrace
1. reagent and instrument
Conventional abdominal operation apparatus (Chinese Shanghai); SERIE7200 respirator (U.S.); Monitor (U.S.); SIMENZ ultrasonic machine (U.S.); The ultrasonic probe L5-12(U.S.); Puncture needle (U.S.); Optical microscope (Japan); Blood automatic biochemical analyzer (Germany); TS-12F automatic water extracter for biological tissue (Hubei China); Roasting sheet machine (Hubei China) LEICA RM2016 paraffin slicing machine (Chinese Shanghai) the LEICA EG1120 tissue embedding machine (Chinese Shanghai) of CS-IV type stand sheet; KD-BL embedding machine freezing stage (Zhejiang Province, China)
2. laboratory animal
18 of healthy male Landraces, body weight 35～40Kg, is provided and is raised at circadian rhythm, the automatic controlled condition of the room temperature cage that places an order by PLA General Hospital Experimental Animal Center.Refining standard pig feed is (9:00 and 16:00) feeding regularly, freely drinks water, and starts experiment after conforming one week, and water is can't help in art fasting the previous day, and all operations all complete in PLA General Hospital standard animal surgery chamber.
3. feasibility and study on the efficiency
3.1 animal groupings
12 Landraces are divided into two groups at random, 6 every group: hepatic segments excision group (segmental hepatectomy, SH group) regulating liver-QI Partial Resection group (partial hepatectomy, PH group)
3.2 Animal Model
(1) laboratory animal anesthesia: intramuscular injection 10mg/kg ketalar gives induced anesthesia, auricular vein gives 0.5ml/kg1% pentobarbital sodium after setting up path, after tracheal intubation success, connects respirator.Regulate respirator tidal volume 10ml/kg, 16 beats/min of respiratory frequencys, FIO2:0.4, end expiratory positive pressure remains on 4cmH
2o.By monitor monitoring blood oxygen and heart rate for cartridge clip pig tongue, keep blood oxygen more than 95%, 60～100 beats/min of hearts rate.The path of setting up by auricular vein in art gives normal saline and glucose injection liquid make-up is lost and finish the rear 3,200,000 u penicillin venoclysises that give respectively in preoperative and operation.
(2) SH treated animal operation process
According to the hepatic segments anatomical features of Hepar Sus domestica, we all carry out liver III (left outside leaf hypomere), the left inside industry hypomere of IV b(to every experimental animal) section excision.Concrete steps are as follows:
(I) animal adopts dorsal position, and J-shaped otch successively cuts and stops blooding into abdomen below right costal margin, and round ligament of liver is cut off in ligation;
(II) intraoperative ultrasound is popped one's head in the full liver of liver diaphragmatic surface scanning, the branch of portal vein (III) of recognizing III section is selected in ultrasonoscopy the face along branch of portal vein major axis, under ultrasonic guidance, adopt the puncture needle of 16G to enter portal vein, pull out nook closing member, pumpback is injected 5mlP407-ICG solution by threaded syringe through puncture needle after seeing that blood disorder is in fact really in portal vein.
(IV) observes liver diaphragmatic surface and visceral surface change color, according to ischemia line boundary, with electric knife, carries out labelling.
(V) along electric knife mark line clamp method from disconnected liver parenchyma, compared with large vein, gives after reliable ligation from disconnected.Hepatic pedicle near-end ties, and far-end flows out from the visible residual gel of having no progeny, and continues from disconnected liver parenchyma until complete resection target hepatic segments.
The method that (VI) is same, precisely punctures after utilization intraoperative ultrasound identification IV b section branch of portal vein, injects 5mlP407-ICG solution, according to ischemia border, gives complete resection target hepatic segments.
(VII) warm saline 1000ml rinses abdominal cavity, observe residue liver particularly incisxal edge have no any ischemia or congestion around and change, confirm after intraperitoneal is without active hemorrhage that abdomen is closed in layering.
(3) pH treated animal operation process
(I) animal adopts dorsal position, and J-shaped otch successively cuts and stops blooding into abdomen below right costal margin, and round ligament of liver is cut off in ligation;
(II), owing to cannot knowing target liver section boundary, adopts electric knife in the geometric figure of Position Approximate place marking convention.
(III) adopts clamp method from disconnected liver parenchyma along electric knife mark line, compared with large pipeline, gives after ligation from disconnected, and wound surface carefully stops blooding, and completes partially hepatectomized and performs the operation.
(IV) warm saline 1000ml rinses abdominal cavity, confirms after intraperitoneal is without active hemorrhage that abdomen is closed in layering.
The processing of 3.3 postoperative animals
All animals are postoperative sends back to after clear-headed, and single cage is raised, and ad lib water, tightly observes its ordinary circumstance and complication.And carry out routine blood test and liver function, renal function, lipid examination in preoperative, immediate postoperative, four time points blood drawing in postoperative 3 days, postoperative 7 days.Within postoperative 7 days, again open abdomen, observe particularly liver section situation of its liver, find out whether there is ischemia, the complication such as congestion or gallbladder leakage.Intravenous injection potassium chloride is put to death after animal, thoroughly noses into abdominal cavity, thoracic cavity, leave and take the heart, lung, kidney, the large check pathological section of liver row after formalin fix, the row HE pathological examination that dyes.
4. safety research
Numerous documents have shown that P407 has good biocompatibility, and have been applied to cardiovascular surgery field abroad, and ICG is also the developer of extensive use clinically.But this experiment has still selected 6 Landraces that 18% P407-ICG solution is injected respectively to liver parenchyma, hepatic vein and biliary tract, and it has been carried out to safety research.Operative process is as follows:
(1) animal adopts dorsal position, and J-shaped otch successively cuts and stops blooding into abdomen below right costal margin, and round ligament of liver is cut off in ligation;
(2) Retrograde excision gallbladder is injected 3mlP407-ICG solution by cystic duct stump in common bile duct, and ultrasonic confirmation biliary tract is blocked completely.
(3) under ultrasonic guidance, in siphonal lobe and left inside leaf liver parenchyma, inject 5ml solution left respectively.
(4) under ultrasonic guidance respectively left internal lobe and left outside leaf reflux veins inject 5ml solution, the omnidistance ultrasonic probe monitoring hepatic vein inner gel that uses dissolves situation, records dissolution time T (v).
(5) check that abdominal cavity closes abdomen without abnormal rear layering, art finishes after clear-headed sends back to, and single cage is raised, and ad lib water is tightly observed.
(6) within postoperative the 3rd day, choose at random 3 and again open abdomen, detect abdominal cavity situation.
(7) cross-section common bile duct, drive in the wrong direction to inject 3mlP407-ICG solution to bile duct near-end, gelling phenomenon occurs as seen at once and cause biliary tract to stop up, and take and looks at lower bile resurgent straight as standard recording biliary tract logical time T (b) again.
(8) cut left outside leaf, left inside leaf is treated postoperative row pathologic finding, P407-ICG residual condition in liver parenchyma while observing first operation.
(9) intravenous injection potassium chloride is put to death animal, leaves and takes common bile duct, the heart, lung, the capable pathologic finding of kidney.
(10) within postoperative the 7th day, perform the operation for the second time remaining 3 Landraces are capable, operate identical with postoperative the 3rd day.
Choose preoperative, immediate postoperative, postoperative 3 days, postoperative 7 days four time points blood drawing promoting the circulation of blood routines and liver function, renal function, lipid examination, all specimen are dipped into formalin after large pathology section examination all, is convenient to the pathological examination of row HE dyeing in the future.
5. sample disposal
After whole blood specimen (3ml venous blood) extracts, the section of Song Woyuan biochemical investigation at once adopts automatic clinical chemistry analyzer (German Cobas-Mira Plus, Roche) monitoring alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum albumin (ALB), total bilirubin (Tbil), bilirubin direct (Dbil), gamma-glutamyl based transferase (γ-GT), alkali phosphatase (ALP), serum creatinine (Scr), blood urea nitrogen (BUN), triglyceride (TG), T-CHOL (TC), also send clinical laboratory (2ml venous blood) promoting the circulation of blood routine examination erythrocyte (RBC) simultaneously, leukocyte (WBC), neutrophilic granulocyte (NPC), eosinophilic granulocyte (EPC), basophilic granulocyte (BPC), platelet (PLT), hemoglobin (Hb).
All organ-tissues are all used 10% dipped into formalin to fix.Wherein hepatic segments piece of tissue is all carried out serial section perpendicular to its hepatic pedicle blood vessel long axis direction and is drawn materials, every block organization thickness is 0.5cm, by specimen paraffin embedding, wax stone is cut into the thick wax disk(-sc) of 4 μ m, put into 49 ℃ of warm water and launch, drag for sheet, dry, put into 75 ℃ of baking oven wax removing 30min left and right, then put into successively dimethylbenzene (1) 5min → dimethylbenzene (2) 5min → anhydrous alcohol 2min → 95% ethanol 2min → 85% ethanol 2min dewaxing and put water.Harrsi haematoxylin dyeing 5min left and right → tap water rinses → 75% hydrochloride alcohol differentiation 10 seconds → tap water flushing → warm water anti-blue 5min → tap water and rinses → 95% ethanol 1min → acidify Yihong ethanol dye liquor 2min → tap water flushing → 95% ethanol (1) 1min → 95% ethanol (2) 1min → anhydrous alcohol 1min dimethylbenzene (1) 1min → dimethylbenzene (2) 1min → neutral gum mounting.Tissues observed situation under light microscopic.
6. statistical method
Experimental data represents with mean soil standard deviation (M soil SD).Adopt SPSS13.0 statistical software to do t check, take P<0.05 as diversity remarkable.
7. interpretation of result
(1) feasibility and effectiveness
All animals have all completed operation according to schedule, and in art, zero accident is dead occurs.The postoperative first day walking drinking water of going forward side by side of can standing, after second day, activity freely, is quick on the draw.Wherein SH group operating time (starting to section hemostatic closure from free liver) is compared with group leader PH (144.2 ± 10.2min, 98.3 ± 12.9min; P<0.05), see Fig. 1; But amount of bleeding (is measured: as follows amount of bleeding=gauze weight in wet base+drainage flow-gauze dry weight-flushing normal saline) compared with PH group few (83.3 ± 14.0ml, 119.2 ± 18.6ml; P<0.05), see Fig. 2; When simultaneously secondary is opened abdomen, detect and find that SH group has no gallbladder and leaks (0/6), and PH organizes visible 2 Hepar Sus domestica sections and exists gallbladder to leak (2/6).The postoperative liver function of two treated animals substantially all recovered normal at the 3rd day, postoperative all animal renal functioies, blood fat are showed no extremely, zero difference between group, and wherein postoperative liver function is shown in Fig. 3 and Fig. 4, postoperative all animal renal functioies are shown in Fig. 5 and Fig. 6, and postoperative blood fat is shown in Fig. 7 and Fig. 8.
(2) safety results
The omnidistance detection of intraoperative ultrasound finds that gelling phenomenon occurs the hepatic venous P407-ICG solution of injection at once, dissolves within 15-20min completely; Inject that to dissolve biliary tract completely after the 3ml solution 15min of biliary tract logical again, the liver function of postoperative the 3rd day all animals is recovered normally substantially, has no bilirubin rising etc. extremely, sees Fig. 9 and Figure 10; Inject hepatic parenchymal gel postoperative the 3rd day 2 times when operation pathologic finding absorb completely, biliary tract, liver parenchyma, kidney, heart, lung in the time of postoperative the 3rd day, 7 days pathologic finding be showed no extremely.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a thermosensitive hydrogel compositions, comprises poloxamer block copolymer and indocyanine green, and wherein, the mass ratio of poloxamer block copolymer and indocyanine green is 640: 1～800: 1.
2. thermosensitive hydrogel compositions according to claim 1, is characterized in that, the mass ratio of poloxamer block copolymer and indocyanine green is 720: 1.
3. thermosensitive hydrogel compositions according to claim 1 and 2, is characterized in that, described poloxamer block copolymer is polyoxyethylene-poly-the third ethylene-polyoxyethylene triblock copolymer, and wherein polyoxyethylene is 7: 3 with the mol ratio of poly-the third ethylene.
4. a thermosensitive hydrogel aqueous solution that contains the thermosensitive hydrogel compositions described in any one in claim 1 to 3, wherein, the content of poloxamer block copolymer is 16%～20%(weight/volume).
5. thermosensitive hydrogel aqueous solution according to claim 4, is characterized in that, the content of poloxamer block copolymer is 18%(weight/volume).
6. the thermosensitive hydrogel aqueous solution described in a claim 4 or 5 is in the application for the preparation of in operating labelling blocking-up preparation.
7. application according to claim 6, is characterized in that, described surgical operation comprises accurate hepatic segments resection operation or sub-hepatic segments resection operation.
8. one kind for preparing the test kit of the thermosensitive hydrogel aqueous solution described in claim 4 or 5, it comprises lid (1), box body (2) and is arranged at the inner pad (3) of box body (2), pad (3) surface is provided with four cavitys, wherein, the first reagent bottle (101) containing poloxamer block copolymer is housed in the first cavity (11), the second reagent bottle (102), the 3rd cavity (13) that in the second cavity (12), are equipped with containing indocyanine green are equipped with solution preparation device (103), and the 4th cavity (14) is equipped with syringe (104).
9. test kit according to claim 8, is characterized in that, in described the first reagent bottle (101), in contained poloxamer block copolymer and the second reagent bottle (102), the mass ratio of contained indocyanine green is 640: 1～800: 1, is preferably 720: 1.
10. test kit according to claim 8 or claim 9, it is characterized in that, solution preparation device (103) is graduated bottle, and its scale capacity is counted 5～6.25ml/g with the quality of contained poloxamer block copolymer in the first reagent bottle (101), is preferably 5.56ml/g.
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