CN101569302A - isolated liver perfusion preservation solution - Google Patents

Abstract

The invention discloses an isolated liver perfusion preservation solution, which contains various electrolytes, amino acids, vitamins, choline chloride, glucose, adenosine triphosphate, adenosine, plasma albumin, low molecular weight dextran, red blood cells, verapamil, Dexamethasone, glutathione, allopurinol, ulinastatin, soluble TNF-α and soluble IL-I and other components. The preservation solution of the present invention can not only provide sufficient oxygen, but also provide sufficient energy supply, so it can be effectively applied to the preservation of the isolated liver at 0-4°C, and has the advantages of low production cost, appropriate viscosity, and long perfusion preservation time , less damage to the liver and fewer complications; it can also prevent ischemic injury, cell edema and secondary damage caused by low temperature, and improve the survival rate of the graft in the host; in addition, it can effectively reduce the risk of organ transplantation. Host-versus-graft and graft-versus-host reactions prolong graft survival.

Description

Preservative fluid for perfusing extracorporeal liver

Technical field

The present invention relates to a kind of isolated organ perfusion and preserve liquid, relate in particular to a kind of preservative fluid for perfusing extracorporeal liver.

Background technology

Present treat transplant organ preserve liquid the most frequently used be that UW liquid, HTK preserve liquid, Celsior preserves liquid etc.; the application of two kinds of preservation liquid in spare-part surgery is comparatively extensive before outstanding; these preservation liquid have advantage separately; can protect transplant organ largely; but their preparation cost is higher, expense is expensive; be not easy to transportation, store and use, and the complication of initiation internal organs is quite a few.There is following shortcoming in the liquid of clinical discovery UW preservation at present: cause that higher ischemic type biliary tract damages incidence; Contain the high molecular weight components HES, high viscosity, can not fully pour into each sinus hepaticus, the shortcoming that causes disturbance of circulation also therefore to influence transplanting back graft function .HTK preservation liquid is and UW preserves the liquid phase ratio, if the holding time surpasses 10h, former the non-functional incidence of graft can raise significantly.The clinical application effect that Celsior preserves liquid also needs further research.And biliary complications and liver microcirculation disorders these liquid often are recurrent in perfusion and preservation internal organs.

In addition, in liver transplant, also have a lot of tree-removers can since the holding time restriction lose, even transfer operation is very successful, but because the preservation process causes decline of liver quality and immunological rejection to cause graft failure to the damage of hepatic tissue.Reason that holding time is short and present simple stored refrigerated mode, preservation liquid composition and perfusion situation have direct relation.The characteristics of simple refrigeration are by providing low temperature environment (0～4 ℃) to reduce the organ metabolic rate, and adopt the preservation liquid of hyperosmosis to prevent the hepatic tissue oedema.

Summary of the invention

The technical issues that need to address of the present invention provide a kind of new preservative fluid for perfusing extracorporeal liver, thereby can more fully provide oxygen and energy for liver organization, minimizing is to the infringement of liver, prevent ischemia injury, edema and secondary injury thereof that low temperature causes, improve the survival rate of graft in the host, prolong the time-to-live of graft.

In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:

A kind of preservative fluid for perfusing extracorporeal liver is preserved each component that can contain following content in the liquid for every liter: NaCl 4～8g, KCl 0.1～0.8g, NaH ₂PO ₄H ₂O 20～300mg, MgSO ₄10～200mg, NaHCO ₃2～9g, L-arginine 50～200mg, histidine 15～120mg, L-cystine 25～140mg, L-methionine 5～100mg, l-glutamine 250～1000mg, L-lysine 50～300mg, glycine 5～100mg, L-isoleucine 35～300mg, L-serine 15～100mg, L-leucine 35～300mg, L-tryptophan 5～40mg, L-trorsine 14 0～300mg, L-valine 35～300mg, L-threonine 40～300mg, Choline Chloride 1～16mg, Cobastab ₂0.3～6mg, pantothenic acid 1～16mg, folic acid 1～16mg, pyridoxal 1～20mg, vitamin PP 1～20mg, glucose 0.5～20g, ATP (adenosine triphosphate) 0.1～10g, adenosine 0.5～20g, plasma albumin 5～100g, D-40 5～100g, red blood cell 0.5～300g, Verapamil 0.5～10mg, dexamethasone 2～20mg, glutathione 2～20mg, allopurinol 2～20mg, UTI 25,000～50,000u/kg, and be added into 1 liter of deionized water of preserving liquid measure.

Preferably, the present invention preserves each component that can contain following content in the liquid for every liter: NaCl5.0～7.0g, KCl 0.3～0.5g, NaH ₂PO ₄H ₂O 100～150mg, MgSO ₄50～100mg, NaHCO ₃4.0～5.0g, L-arginine 80～90mg, histidine 40～60mg, L-cystine 50～70mg, L-methionine 30～50mg, l-glutamine 600～800mg, L-lysine 100～200mg, glycine 20～50mg, L-isoleucine 70～100mg, L-serine 40～100mg, L-leucine 80～150mg, L-tryptophan 10～20mg, L-tyrosine 80～150mg, L-valine 80～150mg, L-threonine 100～200mg, Choline Chloride 2～8mg, Cobastab ₂0.5～2.5mg, pantothenic acid 2～8mg, folic acid 3～10mg, pyridoxal 3～10mg, vitamin PP 5～10mg, glucose 4～10g, ATP (adenosine triphosphate) 3～5g, adenosine 2～10g, plasma albumin 20～50g, Dextran-20～50g, red blood cell 50～100g, Verapamil 1～5mg, dexamethasone 4～10mg, glutathione 4～10mg, allopurinol 4～10mg and UTI 25,000～40,000u/kg.

More preferably, the present invention preserves each component that can contain following content in the liquid for every liter: NaCl5.3～6.3g, KCl 0.31～0.50g, NaH ₂PO ₄H ₂O 110～145mg, MgSO ₄58～80mg, NaHCO ₃4.1～4.9g, L-arginine 81～85mg, histidine 48～58mg, L-cystine 51～65mg, L-methionine 34～47mg, l-glutamine 612～700mg, L-lysine 112～154mg, glycine 28～43m g, L-isoleucine 71～92mg, L-serine 41～91mg, L-leucine 82～131mg, L-tryptophan 13～19mg, L-tyrosine 85～143mg, L-valine 86～125mg, L-threonine 133～159mg, Choline Chloride 3～5mg, Cobastab ₂0.9～2.3mg, pantothenic acid 3～4mg, folic acid 4～9mg, pyridoxal 4～9mg, vitamin PP 7～8mg, glucose 4.6～9.9g, ATP (adenosine triphosphate) 3.4～4.9g, adenosine 3～9g, plasma albumin 24～48g, D-40 28～43g, red blood cell 57～71g, Verapamil 1～5mg, dexamethasone 6～7mg, glutathione 6～7mg, allopurinol 4～9mg and UTI 25,000～40,000u/kg.

Preferably, can also add soluble TNF-α 30-300ug/L and/or solubility IL-I 30-300ug/L in the above-mentioned preservative fluid for perfusing extracorporeal liver of the present invention with HVG or graft-versus-host reaction in the middle of the reduction organ transplant process, thereby improve the graft survival rate.

It is to preserve on the basis of liquid, Celsior preservation formula of liquid at multianalysis UW liquid, HTK that the present invention preserves liquid, new development in conjunction with Organ Preservation, simplify the composition of having improved above-mentioned preservation liquid, through formulated repeatedly, it is as follows to compare improvements and be the content and the role that add each component materials:

1, main improvements

(1) with UW liquid phase ratio, do not adopt kapok sugar and HES etc. to increase the composition of liquid viscosity, thereby reduced residual in biliary tract and blood vessel of preservation liquid effectively, avoided causing thus to the infringement of liver organization and the generation of complication.

(2) this pH value (7.35), osmotic concentration (350mosm/L) of preserving liquid is comparatively suitable, (the pH value is 7.4 than UW liquid, osmotic concentration is 350mosm/L), HTK preserves liquid (the pH value is 7.2, osmotic concentration is 310mosm/L), Celsior preserves the easier incidence that reduces the liver cell oedema of liquid (pH value is 7.3, and osmotic concentration is 320mosm/L).

(3) add soluble TNF-α 30-300ug/L and/or solubility IL-I 30-300ug/L and can reduce central HVG of organ transplant process or graft-versus-host reaction, thereby improve the graft survival rate.

2, other improvements:

(1) plasma albumin can be kept colloid osmotic pressure; D-40 can improve CBF, reach to keep the plasma colloid osmotic pressure purpose, and it has better action to microcirculation, and platelet aggregation is also eliminated in prevention, helps pouring into again.

(2) adding erythrocytic purpose is to increase to take oxygen, guarantees effective oxygen carrying content of perfusion preservation liquid; Add energy matter,, fully guarantee the supply of stripped liver utilizable energy quantity of material as glucose, ATP, adenosine etc.; Vitamin b3 (Cobastab ₂), vitamin substances such as folic acid, pyridoxal, pantothenic acid, can give the required coenzyme of analytic metabolism etc.

(3) Verapamil is a calcium antagonist, can alleviate calcium overload, protective wire plastochondria, reduce the oxygen radical generation, and in addition, it still has the effect that suppresses platelet aggregation; Dexamethasone is a membrane stabilizer, has the membrane stability effect.

(4) UTI can play the effect that suppresses the histocyte metabolism, makes the organ that is saved reduce metabolic enzyme under the normal temperature storage temperature, reduces katabolism, prolongs histocyte life.

Preservative fluid for perfusing extracorporeal liver of the present invention is the same with existing preservation liquid to be applicable to 0～4 ℃ of low temperature environment, compared with prior art, the invention has the beneficial effects as follows: not only can provide sufficient oxygen, and can provide sufficient energy to supply with, thereby the low temperature that can be effectively applied to stripped liver is preserved, it is low to have production cost, low viscosity, holding time is longer, little and the few characteristics of complication to liver damage, can effectively reduce HVG and graft-versus-host reaction in the organ transplant process, prolong the time-to-live of graft, improve the survival rate of graft in the host, in addition, can also prevent the ischemia injury that low temperature causes, edema and secondary injury thereof.

Description of drawings

Fig. 1 is that the Pathomorphology of three groups of hepatic tissues phase when 40h changes (HE * 400), and wherein A: embodiment is 2 groups, B:UW liquid group, C:HTK liquid group.

Embodiment

Further specify the present invention with embodiment below, but the present invention is not limited.

Each component materials can both be on sale on market in the following example.

Below the total amount of preservation liquid of every example be 1 liter:

Composition	Embodiment 1	Embodiment 2	Embodiment 3
				NaCl(g)	5.3	6.3	5.3
KCl(mg)	310	450	500
				NaH 2PO 4·H 2O(mg)	110	145	127
MgSO 4(mg)	68	58	80
				NaHCO 3(g)	4.1	4.9	4.4
L-arginine (mg)	82	81	85
				Histidine (mg)	49	48	58
L-cystine (mg)	51	63	65
				L-methionine (mg)	47	34	39
L-glutamine (mg)	700	689	612
				L-lysine (mg)	112	154	151
Glycine (mg)	43	42	28
				L-isoleucine (mg)	71	92	89
L-serine (mg)	41	43	91
				L-leucine (mg)	131	91	82
L-tryptophan (mg)	13	17	19
				L-tyrosine (mg)	143	85	98
L-valine (rag)	86	125	97
				L-threonine (mg)	133	135	159

Choline Chloride (mg)	5	3	3
				Cobastab 2(mg)	2.3	1.8	0.9
Pantothenic acid (mg)	4	3	4
				Folic acid (mg)	4	9	5
Pyridoxal (mg)	4	5	9
				Vitamin PP (mg)	8	7	7
Glucose (mg)	9500	9900	4600
				ATP(g)	4.9	3.4	4.8
Adenosine (g)	9	3	5
				Plasma albumin (g)	46	24	48
D-40 (g)	28	43	32
				Red blood cell (g)	71	65	57
Verapamil (mg)	3	5	1
				Dexamethasone (mg)	6	6	7
Glutathione (mg)	6	7	6
				Allopurinol (mg)	4	9	7
TNF-α(ug/L)	30	200	300
				IL-I(ug/L)	30	200	300
UTI (u/kg)	25000	30000	40000
				Deionized water	Be added into 1 liter	Be added into 1 liter	Be added into 1 liter

Compound method of the present invention: get the part deionized water, with electrolyte (NaCl, KCl, NaH ₂PO ₄H ₂O, MgSO ₄, NaHCO ₃) at first add wherein, add compositions such as each seed amino acid and plasma albumin then, add red blood cell at last, be settled to 1 liter of total amount with deionized water again.

Experimental example 1

1, main material and reagent: laboratory animal is 90 of adult healthy male and female SD rats, about 300g, is divided into embodiment 2 (patent liquid) group, UW liquid group and HTK group at random.Every group is further divided into 3 groups (n=6) according to the holding time (8h, 16h, 24h, 32h, 40h).Main agents is adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenylate (AMP) sodium salt, Proteinase K, UW liquid and HTK liquid and TUNEL method Apoptosis kit etc.

2, method: it is mainly according to list of references [Xu Dongbo that the stripped liver of (1) rat low temperature continues perfusion preservation Preparation of model, Du Zhi, Potts D J, Deng. the foundation of isolated rat liver re-perfusion model (IPRI) and the meaning [J] in the experiment of liver transfer operation organ preservative fluid thereof. biomedical engineering and clinical, 2001,5:1-3.] the biological isolated rat liver re-perfusion model (IPRI) introduced carries out.(2) mensuration of stripped liver organization energy matter: mainly according to list of references [Zhou Zhihua, Cui Xingang, Han Qiucheng, Deng. the how dirty organ preservative fluid in Shanghai is preserved the experimental study [J] of isolated rat liver. the The 2nd Army Medical College journal, 2007,28 (2): 122-126.] the quick isoconcentration high-efficient liquid phase chromatogram technology of introducing detects the content of ATP, ADP, AMP in the different holding time hepatic tissues, and calculates adenosine phosphate total amount TAN=ATP+ADP+AMP.(3) light microscopic is observed the hepatic tissue morphological change that exsomatizes down.(4) the TUNEL method detects the Apoptosis change.(5) statistical procedures: SPSS 11.0 softwares carry out data statistic analysis, and continuous data is represented with x ± s, relatively adopts Student t check between group, and P＜0.05 is variant for statistics.

3, result: put when five of 8h, 16h, 24h, 32h, 40h mutually (1), and each content and adenosine phosphate total amount of organizing ATP, ADP, AMP all reduces, but each content in embodiment 2 (patent liquid) group sees Table 1 apparently higher than other two groups (P＜0.05).(2) om observation result: the liver cell morphology that each group goes up when 8h, 16h mutually is all normal substantially; When 24h, 32h, go up mutually and cellular swelling, steatosis, the visible a spot of neutrophil infiltration in portal area just occur, endochylema is loose, the balloon sample becomes, and visible a small amount of sinusoidal endothelial cell is shed in the sinus hepaticus chamber slight inflammatory reactions such as the generation of no necrosis of liver cells; Mutually above-mentioned inflammation slightly increases the weight of when 40h, the visible neutrophil infiltration in portal area, and the change of liver cell cavity shape, acidophilia or spotty necrosis, but embodiment 2 (patent liquid) group is compared other two groups of inflammation changes light (as Fig. 1).(3) each group is preserved the testing result of liquid to hepatocellular apoptosis; More than in three groups liver cell 8h, 16h, 24h, 32h, 40h each the time all can see the phenomenon of apoptosis mutually, and along with the continuous prolongation of pouring into the holding time, apoptotic cells liquid increases gradually, especially go up more obviously when 32h, 40h mutually, but wherein hepatocellular apoptosis quantity is that embodiment 2 (patent liquid) group is minimum, and HTK liquid group is maximum, UW is mediate, P＜0.05, showing has significant difference, sees Table 2.

Table 1 liver tissues of rats is preserved in the liquid not simultaneously the energy matter content of phase (μ mol/g, n=6, x ± s) at three kinds

Three kinds in table 2 is preserved liquid at the not influence of liver cell apoptotic index relatively simultaneously (n=6, x ± s)

The above results shows, embodiment 2 groups (patent liquid) obviously is better than UW liquid group and HTK liquid group to the effect of isolated rat hepatic energy metabolism, morphosis and hepatocellular apoptosis.

So, the present invention not only can provide sufficient oxygen, and can provide sufficient Power supply, thereby the low temperature that can be effectively applied to stripped liver is preserved, have that production cost is low, viscosity suitably, continue the characteristics of long, little to liver damage and few intercurrent disease of holding time, can effectively reduce HVG and graft-versus-host reaction in the organ migration process, prolong the time-to-live of graft, improve the survival rate of graft in the host, in addition, can also prevent ischemia injury, edema and the secondary injury thereof that low temperature causes. The present invention is not limited only to liver transplant and preserves, and also can be used for the preservation of other organ, tissue and cells of living such as pancreas, kidney.

Claims (7)

1, a kind of preservative fluid for perfusing extracorporeal liver is preserved each component that contains following content in the liquid for every liter: NaCl 4～8g, KCl 0.1～0.8g, NaH ₂PO ₄H ₂O 20～300mg, MgSO ₄10～200mg, NaHCO ₃2～9g, L-arginine 50～200mg, histidine 15～120mg, L-cystine 25～140mg, L-methionine 5～100mg, l-glutamine 250～1000mg, L-lysine 50～300mg, glycine 5～100mg, L-isoleucine 35～300mg, L-serine 15～100mg, L-leucine 35～300mg, L-tryptophan 5～40mg, L-trorsine 14 0～300mg, L-valine 35～300mg, L-threonine 40～300mg, Choline Chloride 1～16mg, Cobastab ₂0.3～6mg, pantothenic acid 1～16mg, folic acid 1～16mg, pyridoxal 1～20mg, vitamin PP 1～20mg, glucose 0.5～20g, adenosine triphosphate 0.1～10g, adenosine 0.5～20g, plasma albumin 5～100g, D-40 5～100g, red blood cell 0.5～300g, Verapamil 0.5～10mg, dexamethasone 2～20mg, glutathione 2～20mg, allopurinol 2～20mg, UTI 25,000～50,000u/kg, and be added into 1 liter of deionized water of preserving liquid measure.

2, preservation liquid as claimed in claim 1 is characterized in that every liter is preserved each component that contains following content in the liquid: NaCl 5.0～7.0g, KCl 0.3～0.5g, NaH ₂PO ₄H ₂O 100～150mg, MgSO ₄50～100mg, NaHCO ₃4.0～5.0g, L-arginine 80～90mg, histidine 40～60mg, L-cystine 50～70mg, L-methionine 30～50mg, l-glutamine 600～800mg, L-lysine 100～200mg, glycine 20～50mg, L-isoleucine 70～100mg, L-serine 40～100mg, L-leucine 80～150mg, L-tryptophan 10～20mg, L-tyrosine 80～150mg, L-valine 80～150mg, L-threonine 100～200mg, Choline Chloride 2～8mg, Cobastab ₂0.5～2.5mg, pantothenic acid 2～8mg, folic acid 3～10mg, pyridoxal 3～10mg, vitamin PP 5～10mg, glucose 4～10g, adenosine triphosphate 3～5g, adenosine 2～10g, plasma albumin 20～50g, Dextran-20～50g, red blood cell 50～100g, Verapamil 1～5mg, dexamethasone 4～10mg, glutathione 4～10mg, allopurinol 4～10mg and UTI 25,000～40,000u/kg.

3, preservation liquid as claimed in claim 1 is characterized in that every liter is preserved each component that contains following content in the liquid: NaCl 5.3～6.3g, KCl 0.31～0.50g, NaH ₂PO ₄H ₂O 110～145mg, MgSO ₄58～80mg, NaHCO ₃4.1～4.9g, L-arginine 81～85mg, histidine 48～58mg, L-cystine 51～65mg, L-methionine 34～47mg, l-glutamine 612～700mg, L-lysine 112～154mg, glycine 28～43mg, L-isoleucine 71～92mg, L-serine 41～91mg, L-leucine 82～131mg, L-tryptophan 13～19mg, L-tyrosine 85～143mg, L-valine 86～125mg, L-threonine 133～159mg, Choline Chloride 3～5mg, Cobastab ₂0.9～2.3mg, pantothenic acid 3～4mg, folic acid 4～9mg, pyridoxal 4～9mg, vitamin PP 7～8mg, glucose 4.6～9.9g, adenosine triphosphate 3.4～4.9g, adenosine 3～9g, plasma albumin 24～48g, D-40 28～43g, red blood cell 57～71g, Verapamil 1～5mg, dexamethasone 6～7mg, glutathione 6～7mg, allopurinol 4～9mg and UTI 25,000～40,000u/kg.

4, preservation liquid as claimed in claim 1 is characterized in that preserving in the liquid for every liter and also contains soluble TNF-α 30-300ug/L and/or solubility IL-I 30-300ug/L.

5, preservation liquid as claimed in claim 4 is characterized in that preserving liquid for every liter is made up of following each component: NaCl 5.3g, KCl 310mg, NaH ₂PO ₄H ₂O 110mg, MgSO ₄68mg, NaHCO ₃4.1g, L-arginine 82mg, histidine 49mg, L-cystine 51mg, L-methionine 47mg, l-glutamine 700mg, L-lysine 112mg, glycine 43mg, L-isoleucine 71mg, L-serine 41mg, L-leucine 131mg, L-tryptophan 13mg, L-tyrosine 143mg, L-valine 86mg, L-threonine 133mg, Choline Chloride 5mg, Cobastab ₂2.3mg, pantothenic acid 4mg, folic acid 4mg, pyridoxal 4mg, vitamin PP 8mg, glucose 9500mg, adenosine triphosphate 4.9g, adenosine 9g, plasma albumin 46g, D-40 28g, red blood cell 710g, Verapamil 3mg, dexamethasone 6mg, glutathione 6mg, allopurinol 4mg, UTI 25000u/kg, soluble TNF-α 30ug/L, solubility IL-I 30ug/L, surplus is a deionized water.

6, preservation liquid as claimed in claim 4 is characterized in that preserving liquid for every liter is made up of following each component: NaCl 6.3g, KCl 450mg, NaH ₂PO ₄H ₂O 145mg, MgSO ₄58mg, NaHCO ₃4.9g, L-arginine 81mg, histidine 48mg, L-cystine 63mg, L-methionine 34mg, l-glutamine 689mg, L-lysine 154mg, glycine 42mg, L-isoleucine 92mg, L-serine 43mg, L-leucine 910mg, L-tryptophan 17mg, L-tyrosine 85mg, L-valine 125mg, L-threonine 135mg, Choline Chloride 3mg, Cobastab ₂1.8mg, pantothenic acid 3mg, folic acid 9mg, pyridoxal 5mg, vitamin PP 7mg, glucose 9900mg, adenosine triphosphate 3.4g, adenosine 3g, plasma albumin 24g, D-40 43g, red blood cell 65g, Verapamil 5mg, dexamethasone 6mg, glutathione 7mg, allopurinol 9mg, UTI 30000u/kg, soluble T NE-α 200ug/L, solubility IL-I 200ug/L, surplus is a deionized water.

7, preservation liquid as claimed in claim 4 is characterized in that preserving liquid for every liter is made up of following each component: NaCl 5.3g, KCl 500mg, NaH ₂PO ₄H ₂O 127mg, MgSO ₄80mg, NaHCO ₃4.4g, L-arginine 85mg, histidine 58mg, L-cystine 65mg, L-methionine 39mg, l-glutamine 612mg, L-lysine 151mg, glycine 28mg, L-isoleucine 89mg, L-serine 91mg, L-leucine 82mg, L-tryptophan 19mg, L-tyrosine 98mg, L-valine 97mg, L-threonine 159mg, Choline Chloride 3mg, Cobastab ₂0.9mg, pantothenic acid 4mg, folic acid 5mg, pyridoxal 9mg, vitamin PP 7mg, glucose 4600mg, adenosine triphosphate 4.8g, adenosine 5g, plasma albumin 48g, D-40 32g, red blood cell 57g, Verapamil 1mg, dexamethasone 7mg, glutathione 6mg, allopurinol 7mg, UTI 40000u/kg, soluble TNF-α 300ug/L, solubility IL-I 300ug/L, surplus is a deionized water.

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