CN103566413B - Thermo-sensitive gel composition and application thereof - Google Patents
Thermo-sensitive gel composition and application thereof Download PDFInfo
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- CN103566413B CN103566413B CN201310522096.3A CN201310522096A CN103566413B CN 103566413 B CN103566413 B CN 103566413B CN 201310522096 A CN201310522096 A CN 201310522096A CN 103566413 B CN103566413 B CN 103566413B
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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
Current surgical operation enters " precisely " epoch, and desirable RESECTION OF LIVER CANCER not only should complete removal of lesions, also should this hepatic segments at removal of lesions place.But the liver of human body is one piece of overall substantial viscera in appearance, 8 sections can be divided into artificially according to the distribution of its internal blood vessel and supply of blood flow, the key realizing accurate hepatic segments/sub-Hepatic segmentectomy is demarcated target hepatic segments or sub-liver section boundary in Rhizoma Atractylodis Macrocephalae, in order to divide a certain liver section boundary in operation process accurately, current clinical conventional method has following three kinds: (1) is by following the trail of hepatic vein in conjunction with liver surface anatomic landmark and intraoperative ultrasound and branch of portal vein decides target liver section boundary in the projection on liver surface.The method is simple to operate, but requires to have enough knowledge to dissecting in liver, and skillfully grasps intraoperative ultrasound.Often adopting the shape that the specimen of excising in this way is all rule, is not section truly or the excision of sub-section, only reaches degree relatively accurately; (2) by dissecting porta hepatis, and being blocked dissect the Glisson base of a fruit of free target hepatic segments in liver parenchyma after, realizing blocking completely after target hepatic segments blood supplies, realizing Hepatic segmentectomy along ischemic boundary.But realize porta hepatis dissect and in liver parenchyma the Glisson base of a fruit of free section comparatively difficult, technical requirement is high, and risk is comparatively large, limits 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.Inject methylene blue after intraoperative ultrasound Conducted Puncture branch of portal vein, there is blue dye in target hepatic segments surface immediately, can complete hepatic segments/sub-Hepatic segmentectomy along indigo plant dye border.Though the method blemish in an otherwise perfect thing is to achieve the target of precisely demarcating hepatic segments border, but from mostly still needing during disconnected liver parenchyma to adopt Pringle method to block porta hepatis, unavoidably ischemical reperfusion injury is caused to liver.Said method none there is not the shortcomings such as complicated operation, risk are larger or expensive, remain not accurately " local is excavated " when therefore a lot of surgeon carries out hepatectomy at present.
Therefore, current Problems existing needs research and development one to have good biocompatibility, have no side effect, can temporary interruption target hepatic segments portal venous flow, and the markup surgical operation preparation of the Ink vessel transfusing realizing 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, provide a kind of thermo-sensitive gel composition be made up of poloxamer block copolymer and indocyanine green, the thermo-sensitive gel composition solution obtained by this thermo-sensitive gel composition has the feature of temperature sensitivity, reversible, nontoxic and labelling, can play for surgical operation and well block and labelling effect, surgeon can be helped to complete the operation of more simple, safer Hepatic segmentectomy accurately.
Present invention also offers a kind of above-mentioned thermosensitive hydrogel aqueous solution for the preparation of the application in operating markup blocking-up preparation.
For convenience of using, the present invention still further provides the test kit containing thermo-sensitive gel composition.
For this reason, first aspect present invention provides a kind of thermo-sensitive gel composition, comprises poloxamer block copolymer and indocyanine green, and wherein, the mass ratio of poloxamer block copolymer and indocyanine green is 640: 1 ~ 800: 1.The mass ratio of preferred poloxamer block copolymer and indocyanine green is 720: 1.
According to the present invention, described poloxamer block copolymer is polyoxyethylene-poly-third ethylene-polyoxyethylene triblock copolymer, and wherein polyoxyethylene is 7: 3 with the mol ratio of poly-third ethylene.Such as, described poloxamer block copolymer is Poloxamer407 (poloxamer188), and its mean molecule quantity is 12000 dalton.
Second aspect present invention provides a kind of thermosensitive hydrogel aqueous solution containing above-mentioned thermo-sensitive gel composition, and wherein, the content of poloxamer block copolymer is 16% ~ 20% (weight/volume).The content of preferred 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, when more than CMC (critical micelle concentration) and CMT (critical micell temperature), 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 continue to heat up, micelle ordered arrangement forms solid gel.And lower than the self-dissolving formation solution again of poloxamer block copolymer time CMC (critical micelle concentration) and CMT (critical micell temperature).
Such as, poloxamer188 (Poloxamer407) also has reversible temperature-sensing property.Compared with other several poloxamers, the concentration that poloxamer188 forms gel is lower, and the concentration forming gel when 25 DEG C 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, be in a liquid state when Poloxamer407 aqueous solution has low temperature, temperature raises and namely changes solid gel into, again transfers liquid reversible temperature-sensing property to when temperature reduces.Further, confirm that it biochemical reaction does not occur in vivo through clinical trial, can through urine with original form metabolism, to human non-toxic's side effect.But this Poloxamer407 aqueous solution is water white, does not possess the effect of labelling or instruction in operation process, the precise positioning of performing the operation cannot be realized.
Based on the above-mentioned characteristic of poloxamer block copolymer, the present inventor attempts Poloxamer407 to be used for surgical operation as a kind of reversible block agent, for making the function of its performance having labelling concurrently and then realization precise positioning and blocking-up in operation, indocyanine green is attempted to add Poloxamer407 aqueous solution, but 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 the critical micell temperature studying P407-ICG thermosensitive hydrogel aqueous solution is necessary, dissolution characteristics, stability, the results are shown in Table 1:
The characteristic of table 1 Poloxamer407-indocyanine green thermosensitive hydrogel aqueous solution
| Concentration/% (weight/volume) | Critical micell temperature/DEG C |
| 16 | 29 |
| 18 | 25 |
| 20 | 22 |
As can be seen from Table 1, be green transparent solution at ambient temperature when the concentration of this Poloxamer407-indocyanine green thermosensitive hydrogel aqueous solution Poloxamer407 is wherein 18% (weight/volume), when environment temperature reaches more than 25 DEG C, such as under animal and human's body body temperature, just may be transformed into solid phase gel state, and when temperature is lower than animal and human's body body temperature, its again self-dissolving become solution, because concentration is far below 15% needed for gelling (weight/volume) occurs again after it enters blood circulation, so solid gel can not be formed again.Should have no side effect containing Poloxamer407 aqueous solution of indocyanine green, can prototype form through homaluria.Utilize this characteristic, this Poloxamer407 aqueous solution containing indocyanine green can be used for making markup 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 for the preparation of the application in operating markup blocking-up preparation.
In the present invention, described surgical operation comprises the operation of accurate Hepatic segmentectomy or the operation of sub-Hepatic segmentectomy.
By cold cut preparation thermosensitive hydrogel aqueous solution in the present invention, wherein indocyanine green can be added to the water with poloxamer block copolymer simultaneously prepares thermosensitive hydrogel aqueous solution, also can after being mixed with poloxamer block copolymer aqueous solution, then add indocyanine green wherein and mix homogeneously is mixed with thermosensitive hydrogel aqueous solution.
Prepared thermosensitive hydrogel aqueous solution or poloxamer block copolymer aqueous solution are placed under cryogenic conditions and use after cold preservation.Described cryogenic conditions is 2 ~ 6 DEG C, is preferably 4 DEG C.The time of described cold preservation is 5 ~ 10 minutes, is preferably 5 minutes.
In one embodiment of the invention, when above-mentioned markup blocking-up preparation is injected (body temperature 37 DEG C) in portal vein in rats, its gelling at once and become solid-state realize labelling and blocking-up in art, after about 10 minutes, it is in Ink vessel transfusing and spontaneous dissolving, because concentration is far below the concentration 15% (weight/volume) again occurred needed for gelling after the solution formed enters blood circulation, so solid phase gel can not be formed again.There is not any biochemical reaction in vivo in this product, has good biocompatibility, have no side effect, finally with prototype form through homaluria.Simultaneously because from as green, be convenient to very much identification in operation, precise positioning of performing the operation can be realized, substantially increase the safety of operation.
In another specific embodiment of the present invention, above-mentioned markup blocking-up preparation is used in the Hepatic segmentectomy operation of Landrace: use ultrasonic guidance that above-mentioned markup blocking-up preparation is injected the blood vessel of target hepatic segments, this above-mentioned markup blocking-up preparation is become solid-state at once from liquid state, block blood flow, ischemic boundary can be seen in corresponding hepatic segments surface, can complete Hepatic segmentectomy operation accurately immediately; From in disconnected liver parenchyma process, because vascular inner gel presents green, effectively guide doctor to break the direction of liver, further increase the safety of operation.
For convenience of using, fourth aspect present invention provides a kind of test kit of above-mentioned thermo-sensitive gel composition, it pad comprising lid, box body and be arranged at tray interior, pad surface is provided with four cavitys, wherein, the first cavity is built with the first reagent bottle containing poloxamer block copolymer, and the second cavity is built with the second reagent bottle containing indocyanine green, 3rd cavity is equipped with solution preparation device, and the 4th cavity is equipped with syringe.
According to the present invention, in described first reagent bottle, contained poloxamer block copolymer is 640: 1 ~ 800: 1 with the mass ratio of contained indocyanine green in the second reagent bottle, preferably 720: 1.
In one embodiment of the invention, solution preparation device is graduated bottle, and its scale capacity counts 5 ~ 6.25ml/g with the quality of poloxamer block copolymer contained in the first reagent bottle, is preferably 5.56ml/g.
The poloxamer block copolymer formulated by the gel combination of poloxamer block copolymer of the present invention and indocyanine green and the thermosensitive hydrogel aqueous solution of indocyanine green can make markup blocking-up preparation for realizing the markup blocking-up of performing the operation and precise positioning in surgical operation, substantially increase 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 DEG C, just can be transformed into solid phase gel state, through spontaneous dissolving in about 10 minutes, because concentration is far below 15% needed for gelling (weight/volume) occurs again after it enters blood circulation, new gel can not be formed again.The markup blocking-up preparation made with this thermo-responsive hydro gel composition solution has temperature sensitivity, feature that is reversible and labelling is nontoxic, and particularly this markup blocking-up preparation has no side effect, can prototype form through homaluria.
By the huge advantage that has that this markup blocking-up preparation is used for the treatment of in hepatocarcinoma be: (1) meets the principle of tumor radical cure: the micrometastasis of sending out in tumor and the section that first occurs is excised simultaneously simultaneously, reduce postoperative recurrence; (2) intraoperative hemorrhage is reduced: do not have the large Glisson base of a fruit three anatomical structure in hepatic segments border, intraoperative hemorrhage can be reduced from disconnected liver parenchyma by face, relative depletion of blood area within a jurisdiction; (3) incidence of complications is reduced: due to trunk and bile duct can not be destroyed, thus largely avoided bile leakage after operation, ischemia appears in residual liver or congestion and then cause the generation of the complication such as hepatic insufficiency; (4) risk sending out transfer in hepatoma carcinoma cell art through portal vein adverse current is reduced.
This markup blocking-up preparation is used for surgical operation and can plays well blocking-up and labelling effect.Use it for clinical, while blocking-up, band can be made to excise position and be easy to recognize in art, realize the precise positioning of operation, can amount of bleeding be greatly reduced simultaneously, and avoid gallbladder in art to leak or sick cell migration.Thus, surgeon can be helped to complete more simple, safer resection operation, improve operation safety.
The present composition is loaded in a kind of test kit of particular design, conveniently can store, transport the present composition, simultaneously conveniently the present composition is mixed with markup blocking-up preparation is used 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 group 144.2 ± 10.2min; PH group 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 group 83.3 ± 14.0ml; PH group 119.2 ± 18.6ml; P<0.05.
Fig. 3 is two groups of liver function (glutamate pyruvate transaminase, ALT) change curves in embodiment 2.
Fig. 4 is two groups of liver function (glutamic oxaloacetic transaminase, GOT, AST) change curves in embodiment 2.
Fig. 5 is two groups of renal function (blood urea nitrogen, BUN) change curves in embodiment 2.
Fig. 6 is two groups of renal function (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.
Detailed description of the invention
Describe the present invention in detail below in conjunction with embodiment, these embodiments 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 unless otherwise indicated.
Test kit for preparing thermosensitive hydrogel aqueous solution of the present invention, it pad 3 comprising lid 1, box body 2 and be arranged at box body 2 inside, pad 3 surface is provided with four cavitys, wherein, first cavity 11 is built with the first reagent bottle 101 containing poloxamer block copolymer, second cavity 12 is equipped with solution preparation device 103 built with containing the second reagent bottle 102 of indocyanine green, the 3rd cavity 13, and the 4th cavity 14 is equipped with syringe 104.
In mentioned reagent box, in described first reagent bottle 101, contained poloxamer block copolymer is 640: 1 ~ 800: 1 with the mass ratio of contained indocyanine green in the second reagent bottle 102, preferably 720: 1.
In mentioned reagent box, solution preparation device 103 is thin mouth graduated bottle, and its scale capacity counts 5 ~ 6.25ml/g with the quality of poloxamer block copolymer contained in the first reagent bottle 101, is preferably 5.56ml/g.
In a preferred embodiment, poloxamer block copolymer 18g is contained in the first reagent bottle 101; Containing indocyanine green 25mg in second reagent bottle 102; Solution preparation device 103 is thin mouth graduated bottle, and its scale capacity is 100ml.
In another preferred embodiment, poloxamer block copolymer 9g is contained in the first reagent bottle 101; Containing indocyanine green 12.5mg in second reagent bottle 102; Solution preparation device 103 is thin mouth graduated bottle, and its scale capacity is 50ml.
Adopt mentioned reagent box by cold cut preparation thermosensitive hydrogel aqueous solution, wherein indocyanine green can be added to the water with poloxamer block copolymer simultaneously prepares thermosensitive hydrogel aqueous solution, also can after being mixed with poloxamer block copolymer aqueous solution, then add indocyanine green wherein and mix homogeneously is mixed with thermosensitive hydrogel aqueous solution.
Prepared thermosensitive hydrogel aqueous solution or poloxamer block copolymer aqueous solution are placed under cryogenic conditions and use after cold preservation.Described cryogenic conditions is 2 ~ 6 DEG C, is preferably 4 DEG C.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, BASF Corp. of Germany); 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 (Moor equipment company limited of Britain); Animal surgery operating theater instruments (Shanghai Medical Apparatus and Instruments Factory).
2. laboratory animal
Healthy male SD rat 10, body weight 240-260g, is provided by Military Medical Science Institute's Experimental Animal Center.Ad lib water, 12 hr light, start experiment after adapting to one week.
3. the preparation of Novel temperature-sensitive gel and the investigation of gelation temperature
(1) P407 solution adopts cold process preparation: take 16% (weight/volume), 18% (weight/volume), P407 needed for 20% (weight/volume) three variable concentrations solution respectively, under magnetic agitation, gradation adds and is equipped with in the glass cillin bottle of distilled water, 4 DEG C of ice-water baths stir and make it be uniformly dispersed, adding ICG in the ratio of every 100ml solution 25mg carries out painted, then put 4 DEG C of refrigerator cold-storages to spend the night, make fully swelling being uniformly dispersed of gel obtain 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, punch and insert the precision thermometer that precision is 0.1 DEG C in the middle of the rubber stopper of bottle, the mercury ball of thermometer submerges in gel solution completely.Stir gel solution by magnetic stir bar, make solution internal temperature even, slowly raise bath temperature, programming rate is about 1 DEG C/min simultaneously., glass cillin bottle is constantly tilted 90 ° meanwhile, with solution do not occur flow time temperature for gelation temperature.METHOD FOR CONTINUOUS DETERMINATION 3 times, getting its meansigma methods is gelation temperature.
4. blocking door vein Effect disquisition
(1) animal is fully conformed, preoperative 12 h fast, can't help water.After ether inhalation anesthesia success, rat dorsal position is fixed on wooden operating board, uses iodophor disinfection.Get Median incision on upper abdomen and enter abdomen, otch is about 1.5cm, is pulled open by otch stomach wall, with aseptic cotton carrier, gastrointestinal is dialled caudal ward with Self-made hook to both sides, fully appears hepatic portal structure.
(2) experimental group 9 SD rats are divided into 3 subgroup A, B, C at random, each subgroup 3 animals.Extract P407-ICG solution with 1ml syringe, inject 0.5ml in the main portal vein that accurately punctures is backward, the solution concentration that A, B, C tri-subgroups are injected is 16%, 18%, 20% respectively, observes Xining.Matched group 1 rat adopts vascular clamp to be blocked separately by portal vein.Laser Perfusion Imaging instrument is adopted to monitor the perfusion situation that all rats block front and back liver, in order to observe the barrier effect of each group.
(3) observe main portal vein under direct-view, wait for its lead to again and use use laser Perfusion Imaging instrument to confirm liver perfusion closes abdomen after returning 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 DEG C, in table 2.The postoperative equal long-term surviving of all rats, has no any complication; After experimental group A, B, C and matched group portal venous thrombosis, rat liver pours into and all significantly declines, and laser Perfusion Imaging instrument monitoring display perfusion value decline average out to 144PU, 148PU, 145PU and 131PU, in table 2; Experimental group intrinsic block 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 exceeding Xining, three subgroup rat liver perfusion situations all return to and block front level.
Table 2 variable concentrations P407-ICG solution gel concentration and blocking-up rat portal vein time limit
Can find out, this experiment adopts cold cut to prepare 16%, 18%, the P407-ICG thermosensitive hydrogel aqueous solution of 20% 3 kind of variable concentrations, result shows its gelation temperature and is respectively 29 DEG C, 26 DEG C and 22 DEG C, be injected into (36.5 DEG C) in portal vein in rats to form stable green gel at once 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 blocked is longer, but the P407-ICG solution gel temperature that concentration is 20% is 22 DEG C, this stores to temperature sensitive hydrogel and uses and all brings certain problems, and although liquid (gelation temperature is 29 DEG C) can be kept under the solution room temperature of 16%, but because of the deficiency (only having 5min) that its concentration causes Xining too short close to critical gelation concentration (15%), therefore we select concentration be 18% P407-ICG solution carry out next step large zoopery, so not only solution at room temperature can keep liquid, be convenient to store and use, also blocked ideal duration simultaneously, be convenient to subsequent procedures operation.The equal long-term surviving of rat after this experimentation, have no the exceptions such as any complication, this result is consistent with other bibliographical informations, demonstrates the safety of P407-ICG thermosensitive hydrogel aqueous solution trans-portal vein injection.
Embodiment 2: the Hepatic segmentectomy experiment of Landrace
1. reagent and instrument
General abdominal operating theater instruments (Chinese Shanghai); SERIE 7200 respirator (U.S.); Monitor (U.S.); SIMENZ ultrasonic machine (U.S.); Ultrasonic probe L5-12 (U.S.); Puncture needle (U.S.); Optical microscope (Japan); Blood biochemistry automatic analyzer (Germany); TS-12F automatic water extracter for biological tissue (Hubei China); CS-IV type stand sheet bakes sheet machine (Hubei China) LEICA RM2016 paraffin slicing machine (Chinese Shanghai) LEICA EG1120 tissue embedding machine (Chinese Shanghai); KD-BL embedding machine freezing stage (Zhejiang Province, China)
2. laboratory animal
Healthy male Landrace 18, body weight 35 ~ 40Kg, is provided by PLA General Hospital Experimental Animal Center and raises at circadian rhythm, the automatic controlled condition of the room temperature cage that places an order.Refine standard pig feed timing (9:00 and 16:00) feeding, freely drink water, start experiment after conforming one week, art fasted for one day prior can't help water, and all operations all complete in PLA General Hospital standard animal operating room.
3. feasibility and study on the efficiency
3.1 animal groupings
12 Landraces are divided into two groups at random, often organize 6: Hepatic segmentectomy group (segmental hepatectomy, SH group) and major liver resection group (partial hepatectomy, PH group)
3.2 Animal Model
(1) laboratory animal anesthesia: intramuscular injection 10mg/kg ketalar gives induced anesthesia, and auricular vein gives 0.5ml/kg1% pentobarbital sodium after setting up path, connects respirator after tracheal intubation success.Regulate respirator tidal volume 10ml/kg, respiratory frequency 16 beats/min, FIO2:0.4, end expiratory positive pressure remains on 4cmH
2o.By cartridge clip pig tongue monitor monitoring blood oxygen and heart rate, keep blood oxygen more than 95%, heart rate 60 ~ 100 beats/min.The path set up by auricular vein in art is given normal saline and glucose injection liquid make-up and loses and terminate to give 3,200,000 u penicillin venoclysises respectively afterwards 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 excision of IV b (left inside industry hypomere) section to every experimental animal.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) recognizing III section selects the face along branch of portal vein major axis in ultrasonoscopy, ultrasound guidance adopts the puncture needle of 16G to enter portal vein, pull out nook closing member, pumpback see blood disorder real true in portal vein after, inject 5mlP407-ICG solution by threaded syringe through puncture needle.
(IV) observes liver diaphragmatic surface and the change of visceral surface color, carries out labelling according to ischemia line boundary electric knife.
(V), along electric knife mark line clamp method from disconnected liver parenchyma, comparatively large vein gives after reliable ligation from disconnected.Hepatic pedicle near-end ties, and far-end flows out from 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 using intraoperative ultrasound to recognize IV b section branch of portal vein, injects 5mlP407-ICG solution, give complete resection target hepatic segments according to ischemic boundary.
(VII) warm saline 1000ml rinses abdominal cavity, observes residue liver and particularly has no any ischemia around incisxal edge or congestion changes, confirm that intraperitoneal closes abdomen without layering after active hemorrhage.
(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 know 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, and from breaking after comparatively large pipeline gives ligation, wound surface carefully stops blooding, and completes partially hepatectomized operation.
(IV) warm saline 1000ml rinses abdominal cavity, confirms that intraperitoneal closes abdomen without layering after active hemorrhage.
The process of 3.3 postoperative animals
All animals are postoperative sends back to after clear-headed, and single cage is raised, ad lib water, its ordinary circumstance of close observation and complication.And carry out routine blood test and liver function, renal function, lipid examination in preoperative, immediate postoperative, four time point blood drawings in postoperative 3 days, postoperative 7 days.Within postoperative 7 days, again open abdomen, observe its liver particularly liver section situation, find out whether there is ischemia, the complication such as congestion or gallbladder leakage.Intravenous injection potassium chloride is put to death after animal, and thoroughly nose into abdominal cavity, thoracic cavity, after leaving and taking the heart, lung, kidney, the large check pathological section of liver row, formalin is fixed, and row HE dyes pathological examination.
4. safety research
Numerous document has shown that P407 has good biocompatibility, and has 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 the P407-ICG solution of 18% is injected liver parenchyma, hepatic vein and biliary tract respectively, has carried out safety research to it.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, in common bile duct, inject 3mlP407-ICG solution by cystic duct stump, ultrasonic confirmation biliary tract blocks completely.
(3) ultrasound guidance injects 5ml solution respectively in left outside leaf and left inside leaf liver parenchyma.
(4) ultrasound guidance injects 5ml solution respectively to left inside leaf and left outside leaf reflux veins, and the omnidistance ultrasonic probe monitoring hepatic vein inner gel that uses dissolves situation, record dissolution time T (v).
(5) check that abdomen is closed in abdominal cavity rear layering without exception, art finish clear-headed after send back to, single cage is raised, ad lib water, close observation.
(6) postoperative 3rd day random selecting 3 opens abdomen again, detects abdominal cavity situation.
(7) cross-section common bile duct, drives in the wrong direction to bile duct near-end and injects 3mlP407-ICG solution, gelling phenomenon occurs as seen at once and causes biliary tract to block, to look at time T (b) that lower bile resurgent leads to for standard recording biliary tract again straight.
(8) cut left outside leaf, left inside leaf treats postoperative row pathologic finding, P407-ICG residual condition in liver parenchyma when observing initial surgery.
(9) intravenous injection potassium chloride puts to death animal, leaves and takes common bile duct, the heart, lung, the capable pathologic finding of kidney.
(10) capable for remaining 3 Landraces second time was performed the operation in postoperative 7th day, operate identical with postoperative 3rd day.
Choose preoperative, immediate postoperative, four time points blood drawing promoting the circulation of blood in postoperative 3 days, postoperative 7 days conventional and liver function, renal function, lipid examination, all specimen are dipped into formalin after large pathology section examination all, is convenient to row HE in the future and dyes pathological examination.
5. sample disposal
Whole blood specimen (3ml venous blood) gives my biochemical investigation section of institute to adopt automatic clinical chemistry analyzer (German Cobas-Mira Plus at once after extracting, Roche) alanine aminotransferase (ALT) is monitored, 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 all use 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 4 μm of thick wax disk(-sc)s, put into 49 DEG C of warm water to launch, drag for sheet, dry, put into 75 DEG C of baking oven wax removing about 30min, then put into dimethylbenzene (1) 5min → dimethylbenzene (2) 5min → anhydrous alcohol 2min → 95% ethanol 2min → 85% ethanol 2min dewaxing successively and put water.Harrsi haematoxylin dyeing about 5min → tap water → 75% hydrochloride alcohol breaks up 10 seconds → tap water → warm water anti-blue 5min → tap water → 95% ethanol 1min → acidify Yihong ethanol dye liquor 2min → tap water → 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).Adopting SPSS13.0 statistical software to do t inspection, is that diversity is remarkable with P<0.05.
7. interpretation of result
(1) feasibility and effectiveness
All animals complete operation all according to schedule, and in art, zero accident death occurs.First post-operative day walking of can standing is gone forward side by side drinking water, and after second day, activity freely, is quick on the draw.Wherein SH group operating time (to section hemostatic closure from free liver) comparatively group leader PH (144.2 ± 10.2min, 98.3 ± 12.9min; P<0.05), Fig. 1 is seen; But amount of bleeding (measuring as follows: amount of bleeding=gauze weight in wet base+drainage flow-gauze dry weight-flushing normal saline) is PH group few (83.3 ± 14.0ml, 119.2 ± 18.6ml comparatively; P<0.05), Fig. 2 is seen; Detect when secondary opens abdomen simultaneously and find that SH group has no gallbladder and leaks (0/6), and there is gallbladder leakage (2/6) in visible 2 the Hepar Sus domestica sections of PH group.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 exception, 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
Intraoperative ultrasound whole process detects and finds that injecting hepatic venous P407-ICG solution at once gelling phenomenon occurs, and dissolves within 15-20min completely; Dissolve biliary tract completely after injecting the 3ml solution 15min of biliary tract to lead to, the liver function of postoperative 3rd day all animal recovers normal substantially again, has no the exceptions such as bilirubin rising, sees Fig. 9 and Figure 10; When injecting the postoperative 3rd day 2 times operations of hepatic parenchymal gel, pathologic finding absorbs completely, and when postoperative 3rd day, 7 days of biliary tract, liver parenchyma, kidney, heart, lung, pathologic finding is showed no exception.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. the thermosensitive hydrogel aqueous solution containing thermo-sensitive gel composition, described thermo-sensitive gel composition comprises poloxamer block copolymer and indocyanine green, and wherein, the content of poloxamer block copolymer is 18% (weight/volume); The mass ratio of poloxamer block copolymer and indocyanine green is 640: 1 ~ 800: 1.
2. thermosensitive hydrogel aqueous solution according to claim 1, is characterized in that, the mass ratio of poloxamer block copolymer and indocyanine green is 720: 1.
3. thermosensitive hydrogel aqueous solution according to claim 1 and 2, is characterized in that, described poloxamer block copolymer is polyoxyethylene-poly-third ethylene-polyoxyethylene triblock copolymer, and wherein polyoxyethylene is 7: 3 with the mol ratio of poly-third ethylene.
4. the thermosensitive hydrogel aqueous solution in a claim 1-3 described in any one is for the preparation of the application in operating markup blocking-up preparation.
5. application according to claim 4, is characterized in that, described surgical operation comprises the operation of accurate Hepatic segmentectomy or the operation of sub-Hepatic segmentectomy.
6. one kind for preparing the test kit of the thermosensitive hydrogel aqueous solution in claim 1-3 described in any one, it comprises lid (1), box body (2) and be arranged at the inner pad (3) of box body (2), pad (3) surface is provided with four cavitys, wherein, first cavity (11) is built with the first reagent bottle (101) containing poloxamer block copolymer, second cavity (12) is built with the second reagent bottle (102) containing indocyanine green, 3rd cavity (13) is equipped with solution preparation device (103), 4th cavity (14) is equipped with syringe (104).
7. test kit according to claim 6, is characterized in that, solution preparation device (103) is graduated bottle, and its scale capacity is counted as 5.56ml/g with the quality of poloxamer block copolymer contained in the first reagent bottle (101).
8. the test kit according to claim 6 or 7, it is characterized in that, in described first reagent bottle (101) in contained poloxamer block copolymer and the second reagent bottle (102) contained by the mass ratio of indocyanine green be 640: 1 ~ 800: 1.
9. test kit according to claim 8, is characterized in that, in described first reagent bottle (101) in contained poloxamer block copolymer and the second reagent bottle (102) contained by the mass ratio of indocyanine green be 720: 1.
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